ABSTRACT AUERNIK, KATHRYNE SHERLOCK. Functional Genomics Analysis of Metal Mobilization by the Extremely Thermoacidophilic Archa

ABSTRACT

AUERNIK, KATHRYNE SHERLOCK. Functional Genomics Analysis of Metal Mobilization by the Extremely Thermoacidophilic Archaeon Metallosphaera sedula. (Under the direction of Dr. Robert Kelly.)

Biomining processes recovering base, strategic and precious metals have predominantly utilized mesophilic bacteria, but relatively low yields have impacted wider application of this biotechnology. However, the use of high temperature microorganisms offers great potential to increase metal mobilization rates. Metallosphaera sedula (Mse) is an extremely thermoacidophilic archaeon with bioleaching capabilities, although little is known about the physiology of this microorganism. To better characterize Mse, its genome was sequenced and a whole genome oligonucleotide microarray was constructed for transcriptional response analysis. The physiological and bioenergetic complexities of Mse bioleaching were studied focusing on iron oxidation, sulfur oxidation, and growth modes

(heterotrophy, autotrophy, and mixotrophy). The transcriptomes corresponding to each of these elements were examined for clues to the mechanisms by which Mse oxidizes inorganic energy sources (i.e. metal sulfides) and fixes CO2. Quinol/terminal oxidases important for maintaining intracellular pH and contributing to ATP generation via proton pumping were stimulated by different energy sources. The soxABCDD’L genome locus

(Msed_0285-Msed_0291) was stimulated in the presence of reduced inorganic sulfur compounds (RISCs) and H2, while the soxNL-CbsABA cluster (Msed_0500-Msed_0504) was induced by Fe(II). Two similar copies of the SoxB/CoxI-like cytochrome oxidase subunit, foxAA’ (Msed_0484/Msed_0485) were implicated in fox cluster oxidation of Fe(II), as well as other energy sources. The doxBCE locus (Msed_2030-Msed2032) did not respond uniformly to either Fe(II) or RISCs, but was up-regulated in the presence of chalcopyrite (CuFeS2). A similar response was also observed for a putative rusticyanin

(Msed_0966, rus), thiosulfate: quinone oxidoreductase (Msed_0363/Msed_0364, doxDA), and a putative sulfide:quinone oxidoreductase (Msed_1039, sqr), all three of which are candidates to serve as primary electron acceptors from inorganic substrates. Putative proteins implicated in the generation of reducing equivalents were identified (Dms/Sre-like reductase and Hdr-like reductases). Mixotrophy in Mse was defined as a strong preference for organic carbon combined with concomitant use of multiple inorganic (and organic) energy sources, if available. This growth mode was observed during CuFeS2 bioleaching, with organic carbon most likely obtained via recycling of lysed cell material.

Functional Genomics Analysis of Metal Mobilization by the Extremely Thermoacidophilic Archaeon Metallosphaera sedula

by Kathryne Sherlock Auernik

A dissertation submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the Degree of Doctor of Philosophy

Chemical and Biomolecular Engineering

Raleigh, North Carolina

2009

APPROVED BY:

______David F. Ollis Jason M. Haugh

______Amy M. Grunden Robert M. Kelly Chair of Advisory Committee BIOGRAPHY

Kathryne Sherlock Auernik grew up in a small town outside of Rochester, NY. She attended the University of Notre Dame (Notre Dame, IN) and received a Bachelor of

Science degree in chemical engineering and a certificate in materials science in January

2001. After spending 3.5 years providing technical support for the manufacture of viral vaccines (Merck & Co., Inc., West Point, PA), she matriculated to the graduate program in the department of chemical and biomolecular engineering at North Carolina State University in search of more formalized biotechnology-related coursework and an opportunity to conduct academic research. She obtained a non-thesis Master of Science in chemical engineering in 2006. Following completion of her doctoral degree in chemical and biomolecular engineering and minor in biotechnology in 2009, she plans to return to work providing technical support for the manufacture of viral vaccines (Merck & Co., Inc.,

Durham, NC).

ii TABLE OF CONTENTS

LIST OF TABLES...... v LIST OF FIGURES ...... viii LIFE IN HOT ACID: PATHWAY ANALYSES IN EXTREMELY THERMOACIDOPHILIC ARCHAEA ...... 1 Abstract...... 2 Introduction ...... 2 Extremely thermoacidophilic archaea and their physiological characteristics...... 3 Mechanisms of resistance to and survival in hot acid ...... 4 Molecular genetics of extreme thermoacidophiles ...... 8 Bioleaching...... 10 Other developments...... 13 Summary...... 15 Acknowledgements ...... 15 References...... 15 THE GENOME SEQUENCE OF THE METAL-MOBILIZING, EXTREMELY THERMOACIDOPHILIC ARCHAEON METALLOSPHAERA SEDULA PROVIDES INSIGHTS INTO BIOLEACHING-ASSOCIATED METABOLISM...... 29 Abstract...... 30 Introduction ...... 31 Materials and Methods...... 35 Results and Discussion...... 37 Organization and general features of the genome...... 37 Iron oxidation...... 38 Sulfur oxidation...... 41 Carbon metabolism...... 42 Metal Tolerance...... 44 Adhesion ...... 45 Transcriptional response of M. sedula to FeSO4...... 47 Summary...... 49 Acknowledgements ...... 50 References...... 50 Appendix: Supplementary Materials ...... 65 COMPONENTS OF ELECTRON TRANSPORT CHAINS IN THE EXTREMELY THERMOACIDOPHILIC CRENARCHAEON METALLOSPHAERA SEDULA IDENTIFIED THROUGH IRON AND SULFUR COMPOUND OXIDATION TRANSCRIPTOMES ...... 70 Abstract...... 71 Introduction ...... 72 Materials and Methods...... 74 Results ...... 76 Quinone reduction from organic electron donors ...... 76 Quinone reduction via inorganic electron donors...... 77

iii TABLE OF CONTENTS (continued)

New components of Fe(II)-induced electron transport chains...... 78 New components of RISC-induced electron transport chains...... 78 Quinone oxidation via terminal oxidase complexes ...... 81 Fe(II) and RISC-induced hypothetical proteins ...... 84 Discussion...... 85 Summary...... 90 Acknowledgements ...... 91 References...... 91 Appendix: Supplementary Materials ...... 102 PHYSIOLOGICAL VERSATILITY OF THE EXTREMELY THERMOMACIDOPHILIC ARCHAEON METALLOSPHAERA SEDULA SUPPORTED BY HETEROTROPHY, AUTOTROPHY, AND MIXOTROPHY TRANSCRIPTOMES...... 114 Abstract...... 115 Introduction ...... 116 Materials and Methods...... 118 Results and Discussion...... 120 Growth physiology...... 120 Heterotrophic growth...... 121 Autotrophic growth...... 126 Mixotrophic growth...... 132 Summary...... 135 Acknowledgements ...... 136 References...... 136 Appendix: Supplementary Materials ...... 153 BIOLEACHING TRANSCRIPTOMES OFMETALLOSPHAERA SEDULA REVEAL COMPLEX MIXOTROPHIC PHYSIOLOGY ...... 166 Abstract...... 167 Introduction ...... 168 Materials and Methods...... 169 Results and Discussion...... 172 Growth physiology...... 172 Iron oxidation...... 174 Sulfur oxidation...... 176 Metal Tolerance...... 178 Growth Mode...... 179 Summary...... 181 Acknowledgements ...... 182 References...... 182 Appendix: Supplementary Materials ...... 191

iv LIST OF TABLES

Table 1.1 Selected features of extreme thermoacidophiles with sequenced genomes...... 24

Table 2.1 Transcriptional response of YE-grown M. sedula to ferrous sulfate...... 59

Table 2.A1 ORFs differently expressed 2.0-fold (log2 fold=1.0) or higher...... 66

Table 3.1 Components of respiratory ETC in the order of Sulfolobales ...... 98

Table 3.2 M. sedula genome loci responding to ferrous iron and reduced inorganic sulfur compounds (RISC) ...... 99

Table 3.A1 ORFs implicated in RISC processing ...... 103

Table 3.A2 Blue copper protein differential transcription ...... 103

Table 3.A3 RISC ETC differential transcription (Msed 0810 – 0818) ...... 104

Table 3.A4 Homologs to RISC-induced ORFs (Msed 0810 – 0818) ...... 105

Table 3.A5 RISC ETC differential transcription (Msed 1542 – 1550) ...... 106

Table 3.A6 Sequences similar to RISC-induced ORFs (Msed 1542 - 1550)...... 107

Table 3.A7 Terminal oxidases potentially involved in RISC oxidation ...... 108

Table 3.A8 FOX ETC differential transcription...... 109

Table 3.A9 ORFs with significant differential transcription ...... 111

Table 4.1 Occurrence of select genes in extremely thermoacidophilic genomes...... 141

Table 4.2 ORFs up-regulated on heterotrophy in M. sedula genome ...... 142

Table 4.3 Putative biosynthesis ORFs upregulated on autotrophy in M. sedula genome...... 144

Table 4.4 ORFs up-regulated or down-regulated on mixotrophy in M. sedula genome...... 146

Table 4.A1 Annotated oligo-/di-peptide transporters in M. sedula...... 154

LIST OF TABLES (continued)

Table 4.A2 Putative TCA cycle ORFs (including pyruvate: ferredoxin oxidoreductase-like sequences) demonstrating differential transcription...... 155

Table 4.A3 Carbonic anhydrases annotated in the M. sedula genome ...... 156

Table 4.A4 Potential ORFs involved with CO2 fixation via the 3-hydroxypropionate/4-hydroxybutyrate cycle...... 157

Table 4.A5 Potential ORFs involved with NiFe hydrogenases in M. sedula...... 158

Table 4.A6 Potential ORFs involved with electron transfer from hydrogen and/or sulfur in M. sedula ...... 160

Table 4.A7 Potential hydrogenase ORFs involved with electron transfer from sulfur in M. sedula ...... 161

Table 5.A1 Blue copper protein (Bcp) ORFs potentially involved with bioleaching...... 192

Table 5.A2 ORFs potentially involved with terminal oxidase electron transfer from Fe(II)...... 193

Table 5.A3 ORFs with significant differential transcription ...... 196

Table 5.A4 ORFs implicated in RISC processing ...... 197

Table 5.A5 ORFs potentially involved with terminal oxidase electron transfer from RISCs...... 198

Table 5.A6 ORFs potentially involved with electron transfer from RISCs...... 199

Table 5.A7 Potential ORFs involved with CO2 fixation via the 3-hydroxypropionate/4-hydroxybutyrate cycle...... 201

Table 5.A8 Carbonic anhydrases annotated in the M. sedula genome ...... 202

Table 5.A9 ORFs up-regulated on heterotrophy in M. sedula genome ...... 203

Table 5.A10 Putative biosynthesis ORFs upregulated on autotrophy in M. sedula genome...... 206

vi LIST OF TABLES (continued)

Table 5.A11 ORFs up-regulated or down-regulated on mixotrophy in M. sedula genome...... 209

Table 5.A12 Potential ORFs involved with NiFe hydrogenases in M. sedula...... 212

vii LIST OF FIGURES

Figure 1.1 Unrooted 16S phylogentic tree...... 26

Figure 1.2 Metal resistance mechanisms in extreme thermoacidophiles ...... 27

Figure 1.3 The recently proposed 5th cycle of autotrophic carbon fixation ...... 28

Figure 2.1 Selected sections of a full multiple sequence alignment of known and putative proteins involved in Fe oxidation ...... 60

Figure 2.2 Proposed organization of common elements in terminal oxidase complexes of M. sedula...... 61

Figure 2.3 The 3-hydroxypropionate cycle ...... 62

Figure 2.4 M. sedula attached to a FeS2 substrate ...... 62

Figure 2.5 Volcano plot of M. sedula microarray dye-flip results (YE vs. YE+FeSO4) ...... 63

Figure 2.6 Schematic summarizing Fe oxidation, sulfur metabolism, and adhesion components believed to be important to M. sedula’s biomining capacity...... 64

Figure 3.1 Composite model of respiratory electron transport chain components in the Sulfolobales ...... 100

Figure 3.2 Proposed model of M. sedula respiratory electron transport chain(s)...... 101

Figure 3.A1 Normalized transcription levels (Lsms) of M. sedula blue copper proteins for all conditions tested...... 112

Figure 3.A2 Ratio of foxA/foxA’ transcription levels (Lsm_Msed0484/Lsm_Msed0485) for conditions tested...... 112

Figure 3.A3 Phylogram (constructed using ClustalW2) of DMSO reductase-like sequences (α subunits) labeled with GenBank identifiers...... 113

Figure 3.A4 Phylogram (constructed using ClustalW2) of heterodisulfide reductase-like sequences (HdrB homologs) listed with GenBank identifiers ...... 113

viii LIST OF FIGURES (continued)

Figure 4.1 Growth curves ...... 148

Figure 4.2 Transcriptional levels of M. sedula ORFs implicated in TCA Cycle operation ...... 149

Figure 4.3 Transcriptional levels of M. sedula ORFs implicated in gluconeogenesis ...... 150

Figure 4.4 Transcriptional levels of M. sedula ORFs implicated in the 3- hydroxpropionate/4-hydroxbutyrate cycle ...... 151

Figure 4.5 Transcriptional levels of M. sedula hydrogenase-associated ORFs ...... 152

Figure 4.A1 Alignment (using ClustalW2) of NiFe hydrogenase alpha subunits highlighting 4 Cys Ni-binding motifs ...... 162

Figure 4.A2 Phylogram (using ClustalW2 alignment) of NiFe hydrogenase beta subunits ...... 165

Figure 5.1 Growth curves for M. sedula cultures in the presence of 10g/L chalcopyrite (CuFeS2) and a headspace containing air, 7% CO2 + balance air, or 7% CO2+14% H2 + balance air (line graphs using y-axis at right) ...... 187

Figure 5.2 M. sedula cultures and abiotic controls (ac) in the presence of Chalcopyrite ...... 188

Figure 5.3 Heat plot of electron transfer components involved with oxidation of Fe(II) and RISCs ...... 189

Figure 5.4 Heat plot of ORFs involved in determining M. sedula growth mode on CuFeS2 ...... 190

Chapter 1

Life in hot acid:

Pathway analyses in extremely thermoacidophilic archaea

Kathryne S. Auernik, Charlotte R. Cooper, and Robert M. Kelly*

Department of Chemical and Biomolecular Engineering North Carolina State University Raleigh, NC 27695-7905

Published in Current Opinion in Biotechnology 19(5): 445-453 (2008)

ABSTRACT

The extremely thermoacidophilic archaea are a particularly intriguing group of microorganisms that must simultaneously cope with biologically extreme pHs (≤ 4) and temperatures (Topt ≥ 60°C) in their natural environments. Their expanding biotechnological significance relates to their role in biomining of base and precious metals and their unique mechanisms of survival in hot acid, at both the cellular and biomolecular levels. Recent developments, such as advances in understanding of heavy metal tolerance mechanisms, implementation of a genetic system, and discovery of a new carbon fixation pathway, have been facilitated by availability of genome sequence data and molecular genetic systems. As a result, new insights into the metabolic pathways and physiological features that define extreme thermoacidophily have been obtained, in some cases suggesting prospects for biotechnological opportunities.

INTRODUCTION

Over the past 20 years much has been written about the biotechnological potential of microorganisms from extreme environments, primarily focusing on individual enzymes capable of withstanding the otherwise harsh conditions required for long-term efficacy in bioprocessing environments [1-5]. However, as genome sequence data have become available for extremophiles (The UCSC Archaeal Genome Browser; http://archaea.ucsc.edu) and molecular genetics tools have begun to emerge [6,7], there exists the possibility to go beyond single biocatalytic steps to take advantage of the novel

pathways and physiological characteristics that are intrinsic to these unique microorganisms. By incorporating these features into less extreme organisms and cells and by metabolically engineering extremophiles directly, a new horizon in microbial biotechnology can emerge. Here, we consider the extremely thermoacidophilic archaea, microorganisms that thrive in hot acid.

Extremely thermoacidophilic archaea and their physiological characteristics

For the purposes of this review, an “extreme thermoacidophile” is a microorganism with both an optimal growth temperature ≥ 60oC and an optimal pH of ≤ 4.0. A majority of the extremely thermoacidophilic species studied to date [8] belong to the archaeal orders

Sulfolobales and Thermoplasmatales (Figure 1.1). From what is currently known, it is interesting that the most heat-tolerant extreme thermoacidophiles are not the most acid- tolerant and vice versa. The most thermophilic of the extreme thermoacidophiles,

o o crenarcheon Acidianus infernus, grows at temperatures up to 95 C (Topt of 85-90 C) but at pHs only as low as 1.0 (pHopt 2.0) [9]. In contrast, Picrophilus species of the euryarchaeal order Thermoplasmatales are the most acidophilic, growing at pHs as low as 0 (pHopt 0.7),

o but at temperatures up to only 65°C (Topt of 60 C) [10]. Insights into life in hot acid may be soon forthcoming, since genome sequences exist or are underway for many extreme thermoacidophiles (see Figure 1.1, Table 1.1) (www.genomesonline.org). Furthermore, several new species in known genera of Sulfolobales (Acidianus, Metallosphaera) have been reported, as well as a new member of the Thermoplasmatales, Thermogymnomonas acidicola [11-14]. We may have only scratched the surface with respect to extreme

thermoacidophile diversity, because new environments not previously known to harbor these microorganisms have recently been identified. For example, mathematical modeling and 16S rRNA data suggest that conditions conducive to thermoacidophilic growth exist in deep-sea hydrothermal vents, a hypothesis supported by reports of the first euryarchaeon from the order Deep-sea Hydrothermal Vent Euryarchaeotic 2 (DHVE2), Aciduliprofundum

o boonei [15●●]. Although A. boonei (Topt 70 C) grows best at a pH slightly above 4.0, 16S rRNA indicates that this microorganism comprises 10-15% of selected vent-associated archaeal populations, suggesting that there are extreme thermoacidophiles from these sites yet to be isolated [15].

Mechanisms of resistance to and survival in hot acid

The mechanisms by which microbial life thrives in hot acid have been investigated in some detail in recent years, triggered by the availability of genome sequence data, functional genomics tools, and molecular genetics. While the intrinsic basis for this novel growth physiology is not clear, clues are emerging as to how these microorganisms survive in the face of hot, acidic, and often metal-laden conditions which are typically associated with DNA damage, protein denaturation, and other disruptions in cellular processes.

DNA damage and repair. High temperatures and the potential for cytosol acidification heighten the possibility of DNA damage or modification in extreme thermoacidophiles relative to mesophilic neutrophiles. Thus, clues to DNA damage repair may emerge from examination of this cellular function in hot acid biotopes. It is surprising

that basal mutation rates for extreme thermoacidophiles are not particularly high. For example, Sulfolobus acidocaldarius has a spontaneous mutation rate similar to that of E. coli [16]. Furthermore, when Sulfolobus solfataricus and S. acidocaldarius were exposed to

UV-irradiation, no significant increase in transcription of known DNA repair proteins was noted [17●,18]; however, it is possible that these genes are constitutively transcribed at higher levels than in mesophiles. Following irradiation, aggregates resembling those formed during plasmid-mediated conjugation were found, spurring speculation that Sulfolobus species may use conjugational DNA exchange and homologous recombination to repair mutated DNA [17]. In a related study, S. solfataricus infected with the Sulfolobus spindle- shaped virus (SSV1) exhibited a similar, but heightened, response to UV-induced DNA damage, suggesting that viruses may be an evolutionary component of stress management systems [19]. Another spindle-shaped virus, SSV2 from native host “Sulfolobus islandicus”

REY15/4, sent infected “S. islandicus” REY15A cells into a metabolically inactive state upon encountering unfavorable environmental conditions and then played a role in re-starting metabolic activity once favorable growth conditions emerged [20]. Up-regulation of two S. solfataricus recA/rad51 homologs (radA, SSO0250; radA-like, SSO0777) in response to a

DNA-damaging antibiotic led to discovery of the first regulatory protein involved in archaeal

DNA damage repair (Sta1, SSO0048) [21]. Neither the radA-like SSO0777, its sta1 activator, nor radA itself were induced by UV-irradiation, indicating that the nature of DNA damage may drive the specific type of repair response [21].

Heat shock. Though extreme thermoacidophiles thrive at temperatures up to 95oC, they are still susceptible to thermal stresses such that they exhibit both cold shock and heat

shock responses. Extremely thermoacidophilic archaea react to supraoptimal temperatures in much the same way as other microorganisms [22-24]. Most work to date has focused on the archaeal thermosome, or rosettasome, a heat-shock responsive HSP60-like molecular chaperone that has been implicated in many cellular roles [25]. However, recent efforts have shown that heat shock response in extreme thermoacidophiles is extensive, involving much more than chaperones or other proteins involved in protein refolding. When exponentially growing S. solfataricus was shifted from its growth temperature optimum

(80oC) to 90oC, approximately 1/3 of the transcriptome responded within 5 minutes [26].

Included in this set of genes were many insertion elements and chromosomally encoded toxin-antitoxin (TA) loci - 22 TA pairs and 1 solitary toxin – all from the VapBC family [26].

Chromosomally-encoded TA loci in bacteria are thought to be stress response elements

[27], although the role of these tandem protein complexes in archaea has not been examined. Since PIN domain-containing VapCs, the “toxin” component of TA loci, are putative ribonucleases [28,29], these proteins could play an important role in post- transcriptional regulation in archaea, especially during heat shock.

Metal resistance. Extreme thermoacidophiles have developed mechanisms for tolerating heavy metals that are physiologically toxic to most microorganisms (Figure 1.2).

These mechanisms involve their capacity to recover from metal-induced damage (similar to oxidative stress) [30] and to limit the effective concentration of the toxic metal itself. In some cases, enzymes reduce or oxidize metals to less toxic forms - for example, the mercuric reductase in S. solfataricus reduces soluble intracellular Hg2+ to volatile elemental

Hg0 [31●●]. In other cases, metal chelation or complexation can accomplish the same

objective. In Sulfolobus metallicus, a polyphosphate (polyP)-based mechanism is believed to underlie cellular tolerance to high levels of copper; greater accumulation of polyP granules was observed in S. metallicus (considered to have higher levels of Cu tolerance) compared to S. solfataricus, and granule size was noted to decrease as Cu levels were increased [32●]. Other strategies, however, do not involve metal transformation, direct or indirect, and instead are based on exporting toxic metal ions via P-type ATPases [33].

Evidence to date suggests that multiple systems can operate in parallel in extreme thermoacidophiles to provide cumulative tolerance, with particular strategies useful for multiple metals [31-33]. For example, copper tolerance in Sulfolobus species involves efflux

ATPases in addition to the polyP pathway, but the same ATPases also contribute to cadmium tolerance [32,33]. There may also be some intrinsic redundancy in protecting against heavy metal toxicity. Disruption mutants lacking mercuric reductase and its regulator (merAR) were found to still exhibit some mer operon transcription [31]. In fact, creation of a mutant with a disrupted regulator resulted in increased merA expression and consequently increased Hg2+ tolerance [31], underscoring the importance of understanding the regulation of resistance mechanisms with respect to engineering characteristics for bioprocesses. While traditional acclimation and/or spontaneous mutant generation approaches are still useful, direct genetic manipulation offers the possibility of conferring similar levels of metal tolerance increase in a systematic manner.

Molecular genetics of extreme thermoacidophiles

Versatile genetic systems for extreme thermoacidophiles are a critical need for many reasons. Recombinant expression of genes encoding extreme thermoacidophile proteins in commonly used bacterial hosts can be problematic [34,35], likely reflecting intrinsic differences between archaea and bacteria. Also, molecular genetic systems could provide the basis for investigating biological mechanisms enabling life in hot acid.

Fortunately, promising developments along these lines have been reported, including successful efforts with gene disruption [36,37] and protein tagging [6]. For a comprehensive review of the development of molecular genetics for archaea see [7].

Viruses and plasmids. Virus-based plasmids have been tailored for specific needs to support development of genetics systems in extreme thermoacidophiles. The first virus from an extreme thermoacidophile, SSV1, was isolated over 20 years ago from Sulolobus shibatae (B12) [38] and is the one most developed for use in studying Sulfolobus species

[7]. There are some useful features of this virus for molecular genetics. For example, removal of the integrase gene from SSV1 demonstrated that the integrase protein is essential only for integration into the host genome, and not for virus infection and replication

[39]. This characteristic could be exploited for novel vector development, where non- homologous recombination of the vector into the host genome is undesirable. Recently constructed Sulfolobus-E. coli fusion vectors have potential as easily modifiable genetic elements for extreme thermoacidophiles. The fusion shuttle vector pSSVrt, based on “S. islandicus” REY15/4 pSSVx and E. coli pUC19, accommodated insertion of foreign sequences up to ~11 Kb with efficient propagation and vector stability at high-copy numbers with no integration [40]. Furthermore, the pRN1-E. coli transposon fusion, noted

for its relatively small size (5.4 Kb) and stable copy numbers of 10-20 in mid-log phase, is stable in S. acidocaldarius and S. solfataricus, as well as in E. coli. [41]. By adding the pyrE gene to this plasmid and using pyrE mutants, a selectable marker could be introduced into the host. An SSV1-E. coli pUC18 reporter gene system was developed with selectable marker genes pyrEF, both heat- and arabinose-inducible promoters, and convenient restriction sites [42]. The improved vector (modified from pMJ03) was used for heterologous and homologous production of tagged proteins in S. solfataricus. All of these developments are exciting and offer promise for expanding molecular genetics capabilities in extreme thermoacidophiles.

Gene disruption. Construction of directed gene deletion mutants in extremely thermoacidophilic archaea is a significant challenge, but progress is being made on this front. The focus has been on S. solfataricus PBL2025, a constructed mutant of S. solfataricus 98/2, which lacks about 50 genes, including lacS [43]. Thus, the inability to grow on lactose-based minimal media provides a selectable marker. A protocol for efficient integration of exogenous DNA into the S. solfataricus PBL2025 genome has been described [36]. A similar method was used to construct a deficient mutant to study α- amylase function and regulation in S. solfataricus [37]. Markerless exchange using a plasmid that encodes a cloned copy of a modified DNA sequence and a selectable marker gene, again lacS for natural deletion mutant PBL2025, has also been used for development of mercury reductase (merA) deficient Sulfolobus solfataricus [31].

Recombinant production of extremely thermoacidophilic proteins. Production of extremely thermoacidophilic proteins in mesophilic hosts (e.g., E. coli) can take

advantage of overexpression and simplified purification methods (e.g., heat treatment), but codon usage and inclusion body problems often temper the enthusiasm for this approach

[34,35]. Some solutions to existing problems have been recently proposed. S. solfataricus genes have rare (compared to E. coli) codon clustering at the 5’ transcript end which specifically inhibits target translation. But, this can be relieved by adding rare codon tRNAs

(utilizing strains like BL21(DE3) CodonPlus-RIL or Rosetta(DE3)) or by changing rare codons (via primer design) to those more frequently translated by the host [34]. Yields of active S. tokodaii and P. torridus proteins produced in E. coli (Rosetta(DE3)) were increased by growth and expression at elevated temperatures up to 46oC, where it was hypothesized that protein synthesis was slowed, contributing to an increase in rate of proper folding [35].

Bioleaching

The biomining industry has a longstanding interest in the use of extreme thermoacidophiles for metals recovery from ores [44,45]. These organisms, as is the case with certain mesophilic chemolithotrophic bacteria such as Acidothiobacillus ferrooxidans

[46●], can liberate precious (e.g., gold) and base (e.g., copper) metals trapped in, and as, metal sulfides (e.g., iron pyrite and chalcopyrite) through dissimilatory oxidative processes.

Biological regeneration of Fe3+ from Fe2+ is key to chemical attack of metal sulfides.

However, biooxidation of reduced inorganic sulfur compounds (RISCs) is also important to prevent the accumulation of passivating sulfur compounds on metal surfaces that can limit metal mobilization rates. Extreme thermoacidophiles grow at temperatures where

mesoacidophilic biocatalysts (or contaminants from non-sterile substrates) cannot, and where passivation from RISCs is nearly eliminated, leading to higher effective leaching rates [46]. Efficacy in biomining environments also requires tolerance of high levels of toxic heavy metals as well as the ability to assimilate inorganic carbon, as organic sources can be scarce in this environment. A full complement of the aforementioned desirable traits is not typically resident in a single native microorganism, but may be in a consortium [46].

Alternatively, extreme thermoacidophile genomes [47-49], including that of a biomining organism [50●], can be examined for pathways responsible for conferring these desirable biomining traits. Taken together with the emerging molecular genetic tools for extreme thermoacidophiles, metabolic engineering of biomining organisms with enhanced properties may soon be a reality.

Iron-sulfur oxidation. Several terminal components of respiratory electron transport chains (ETC) in extreme thermoacidophiles have been known for years [51-54].

However, it was recently shown that the relative membrane concentration of these components depends on the electron donating substrate [55], suggesting involvement in dissimilatory Fe2+ and RISC oxidation. Substrate-dependent ETC expression led to identification of a Fe2+ oxidation- (fox) induced gene cluster in the autotrophic biominer, S. metallicus [56●●]. Comparative genomics subsequently revealed that homologs to this gene cluster are also present in Metallosphaera sedula [50] and Sulfolobus tokodaii, the latter of which was not previously shown to have iron-oxidizing capabilities [56]. This cluster contains not only ORFs similar to previously recognized terminal components (foxABCD), but also ferredoxins and other putative iron-sulfur binding proteins typically involved in

electron transfer (foxFGHJ). This implies that more than terminal components, possibly entire ETCs, are differentially expressed in response to certain substrates. Although the sulfur oxygenase reductase (SOR) does not appear to be directly connected to an ETC

[57●], it was also up-regulated in So compared to Fe2+ grown cells [56]. SOR is believed to participate in the early steps of cytoplasmic sulfur oxidation in extremely thermoacidophilic archaea. Surprisingly, M. sedula, a putative sulfur oxidizer [58●], does not encode SOR

[50], indicating that either some extremely thermoacidophilic archaea have alternative (yet unknown) sulfur oxidation enzymes, or that M. sedula has lost (or never possessed) the capacity to oxidize sulfur (similar to S. solfataricus and S. acidocaldarius) [58].

th CO2 fixation. With the identification of a 5 pathway for inorganic carbon fixation

[59●●], new perspectives on autotrophy have emerged. The new cycle model starts with a

2-carbon acetyl-CoA molecule and assimilates two bicarbonates. The key bicarbonate- incorporating enzyme is a heterotrimeric acetyl-/propionyl-CoA carboxylase [60]. One way in which this new pathway is distinguished from the 3-hydroxypropionate cycle is by a putative homotetrameric 4-hydroxybutyryl-CoA dehydratase (Figure 1.3). While activities have been detected for all steps in this pathway [59], only a few enzymes have been characterized biochemically [59-62]. Comparative genomics suggests multiple candidates

(all having similar sequences) for the final enzymatic reactions of the cycle;experimental work is required to identify which of these candidates supports the function of the final three steps of the pathway, involving re-arrangement of a 4-carbon molecule (crotonyl-CoA) prior to splitting into two molecules of acetyl-CoA. Understanding and controlling regulation of

this new inorganic carbon fixation pathway could lead to improvements in bioleaching through enhanced biomass/biocalatyst levels, and even new CO2 sequestration strategies.

OTHER DEVELOPMENTS

ncRNA. Non-coding RNAs (ncRNAs) in extremely thermoacidophilic archaea, particularly in Sulfolobus species [63-66], could be used for transient control of biocatalytic steps in a bioprocess via dosing of small interfering RNA (siRNA). While many ncRNAs are also small RNAs (sRNA) of 60 nucleotides or less, ncRNAs as long as 500 nucleotides have been reported [63]. Most ncRNAs are characterized by a K-turn motif and appear to possess a post-transcriptional modification or regulation (“silencing”) function. Many ncRNAs recognize their targets via full (“anti-sense”) or partial (“antisense-box motif”) complementarity to transposons, other ORFs, or non-coding RNA (rRNA, tRNA, sRNA, etc.) [63,64]. ncRNAs are thought to interact with proteins to influence structure and function. For example, in S. solfataricus, the Rbp18 protein of the 30S ribosomal subunit has been shown to bind free sRNA (in vitro and in vivo) with some degree of selectivity [65].

In S. acidocaldarius, the anti-sense-box motifs (and their location) in sRNA are important for both proper structural complexation with ribosomal core proteins and complex activity [64]. sRNA produced from clustered regularly interspaced short palindromic repeats (CRISPRs) are believed to interact with catalytically active CRISPR-associated sequences (Cas proteins), which are often encoded immediately up- (crenarchaea) or downstream

(euryarchaea) of CRISPRs [63,66]. Some CRISPRs contain non-repetitive spacer sequences with similarity to viruses or genomic ORFs. The Cas proteins may be involved

with adding/removing spacer sequences to/from CRISPR regions (i.e. Cas1, Cas2), processing of long ncRNA to sRNA form (i.e. Cas3, Cas5), and possibly complexing with resulting sRNAs to form a microbial equivalent of the eukaryotic RNA-induced silencing

(RISC) complex (i.e. Cas4) [66,67].

S-layers and extreme thermoacidophile membranes. S-layers of extremely thermoacidophilic archaea are of interest in nanobiotechnology, because their self- assembled periodicity and uniform properties, as well as thermoacid stability, make them attractive for use in ultrafiltration, immobilization matrix, and coating capacities [68]. Most work to date with these S-layers has focused on structure and properties of the purified self- assembled proteins [69]. Models suggest that archaeal S-layers, with their membrane anchors, help maintain cell shape and stabilize the membrane against environment-induced osmotic pressure changes [70]. Study and use of tetraetherlipids themselves have been somewhat hampered due to high costs/low purification yields. However, recent efforts to defray bioleaching costs by processing by-products have resulted in a lower-cost, higher yield purification process for extremely thermoacidophilic tetraetherlipid, calditoglycerocaldarchaeol [71]. The next significant advances in functional understanding may come from studies of the natural environment of S-layers, in which they are bound to the tetraetherlipid cell membrane [69]. A search through GenBank (July 2008) shows that although S-layer domain proteins are annotated in sequenced extreme thermoacidophiles

(COG 1361), genes related to their modification and assembly are not well known/annotated. Future work in this area will most likely begin with analysis of gene neighborhoods (many containing transporters and/or transcriptional regulators).

Manipulation via genetic tools will be invaluable in study of the formation and regulation of these proton influx barriers with long-term potential to produce tunable acid stability.

SUMMARY

Recently available extreme thermoacidophile genome sequences are revealing novel pathways and strategies that contribute to survival in hot, acidic environments. With the emerging availability of molecular genetics for these microorganisms, metabolic engineering efforts to realize biotechnological opportunities are within reach. Also promising is the prospect of finding novel extreme thermoacidophiles in yet untapped acidic niches, such as deep sea hydrothermal biotopes.

ACKNOWLEDGMENTS

KSA and CRC acknowledge NIH T32 Biotechnology Traineeships for support. This work was funded in part by a grant to RMK from the US National Science Foundation.

REFERENCES

1. Adams MW, Perler FB, Kelly RM: Extremozymes: expanding the limits of biocatalysis. Biotechnology (N Y) 1995, 13:662-668.

2. Antranikian G, Vorgias CE, Bertoldo C: Extreme environments as a resource for microorganisms and novel biocatalysts. Adv Biochem Eng Biotechnol 2005, 96:219- 262.

3. Hough DW, Danson MJ: Extremozymes. Curr Opin Chem Biol 1999, 3:39-46.

4. Tindall KR, Kunkel TA: Fidelity of DNA synthesis by the Thermus aquaticus DNA polymerase. Biochemistry 1988, 27:6008-6013.

5. Atomi H: Recent progress towards the application of hyperthermophiles and their enzymes. Curr Opin Chem Biol 2005, 9:166-173.

6. Sowers KR, DasSarma S, Blum PH: Gene Transfer in Archaea. In Methods for General and Molecular Microbiology. Edited by Reddy CA, Beveridge TJ, Breznak JA, Marzluf GA, Schmidt TM: American Society for Microbiology; 2007.

7. Allers T, Mevarech M: Archaeal genetics - the third way. Nat Rev Genet 2005, 6:58- 73.

8. Garrity GM, Lilburn TG, Cole JR, Harrison SH, Euzeby J, Tindall BJ: Taxonomic Outline of the Bacteria and Archaea, Release 7.7. Edited by: Michigan State University Board of Trustees; 2007:6-31.

9. Huber H, Prangishvili D: The Sulfolobales. In The Prokaryotes, edn 3. Edited by Dworkin M, Falkow S, Rosenberg E, Schleifer K, Stackebrandt E: Springer; 2006:23-50.

10. Huber H, Stetter KO: Thermoplasmatales. In The Prokaryotes, edn 3. Edited by Dworkin M, Falkow S, Rosenberg E, Schleifer K, Stackebrandt E: Springer; 2006:101- 112.

11. Plumb JJ, Haddad CM, Gibson JA, Franzmann PD: Acidianus sulfidivorans sp. nov., an extremely acidophilic, thermophilic archaeon isolated from a solfatara on Lihir Island, Papua New Guinea, and emendation of the genus description. Int J Syst Evol Microbiol 2007, 57:1418-1423.

12. Yoshida N, Nakasato M, Ohmura N, Ando A, Saiki H, Ishii M, Igarashi Y: Acidianus manzaensis sp. nov., a novel thermoacidophilic archaeon growing autotrophically 3+ by the oxidation of H2 with the reduction of Fe . Curr Microbiol 2006, 53:406-411.

13. Kozubal M, Macur RE, Korf S, Taylor WP, Ackerman GG, Nagy A, Inskeep WP: Isolation and distribution of a novel iron-oxidizing crenarchaeon from acidic

geothermal springs in Yellowstone National Park. Appl Environ Microbiol 2008, 74:942-949.

14. Itoh T, Yoshikawa N, Takashina T: Thermogymnomonas acidicola gen. nov., sp. nov., a novel thermoacidophilic, cell wall-less archaeon in the order Thermoplasmatales, isolated from a solfataric soil in Hakone, Japan. Int J Syst Evol Microbiol 2007, 57:2557-2561.

15. Reysenbach AL, Liu Y, Banta AB, Beveridge TJ, Kirshtein JD, Schouten S, Tivey MK, Von Damm KL, Voytek MA: A ubiquitous thermoacidophilic archaeon from deep- sea hydrothermal vents. Nature 2006, 442:444-447. ●● Discusses strategy used to isolate a new order of extremely thermoacidophilic organisms in a low pH deep-sea environment.

16. Grogan DW, Carver GT, Drake JW: Genetic fidelity under harsh conditions: analysis of spontaneous mutation in the thermoacidophilic archaeon Sulfolobus acidocaldarius. Proc Natl Acad Sci U S A 2001, 98:7928-7933.

17. Frols S, Gordon PM, Panlilio MA, Duggin IG, Bell SD, Sensen CW, Schleper C: Response of the hyperthermophilic archaeon Sulfolobus solfataricus to UV damage. J Bacteriol 2007, 189:8708-8718. ● Genome-wide transcriptional response analysis of Sso PH1 and Sso PH1 (SSV1) to UV irradiation to elucidate the DNA damage/repair mechanisms.

18. Gotz D, Paytubi S, Munro S, Lundgren M, Bernander R, White M: Responses of hyperthermophilic crenarchaea to UV irradiation. Genome Biology 2007, 8:R220.

19. Frols S, Gordon PM, Panlilio MA, Schleper C, Sensen CW: Elucidating the transcription cycle of the UV-inducible hyperthermophilic archaeal virus SSV1 by DNA microarrays. Virology 2007, 365:48-59.

20. Contursi P, Jensen S, Aucelli T, Rossi M, Bartolucci S, She Q: Characterization of the Sulfolobus host-SSV2 virus interaction. Extremophiles 2006, 10:615-627.

21. Abella M, Rodriguez S, Paytubi S, Campoy S, White MF, Barbe J: The Sulfolobus solfataricus radA paralogue sso0777 is DNA damage inducible and positively regulated by the Sta1 protein. Nucleic Acids Res 2007, 35:6788-6797.

22. Han CJ, Park SH, Kelly RM: Acquired Thermotolerance and Stressed-Phase Growth of the Extremely Thermoacidophilic Archaeon Metallosphaera sedula in Continuous Culture. Appl Environ Microbiol 1997, 63:2391-2396.

23. Trent JD, Osipiuk J, Pinkau T: Acquired thermotolerance and heat shock in the extremely thermophilic archaebacterium Sulfolobus sp. strain B12. J Bacteriol 1990, 172:1478-1484.

24. Peeples TL, Kelly RM: Bioenergetic Response of the Extreme Thermoacidophile Metallosphaera sedula to Thermal and Nutritional Stresses. Appl Environ Microbiol 1995, 61:2314-2321.

25. Kagawa HK, Yaoi T, Brocchieri L, McMillan RA, Alton T, Trent JD: The composition, structure and stability of a group II chaperonin are temperature regulated in a hyperthermophilic archaeon. Mol Microbiol 2003, 48:143-156.

26. Tachdjian S, Kelly RM: Dynamic metabolic adjustments and genome plasticity are implicated in the heat shock response of the extremely thermoacidophilic archaeon Sulfolobus solfataricus. J Bacteriol 2006, 188:4553-4559.

27. Pandey DP, Gerdes K: Toxin-antitoxin loci are highly abundant in free-living but lost from host-associated prokaryotes. Nucleic Acids Res 2005, 33:966-976.

28. Clissold PM, Ponting CP: PIN domains in nonsense-mediated mRNA decay and RNAi. Curr Biol 2000, 10:R888-890.

29. Daines DA, Wu MH, Yuan SY: VapC-1 of nontypeable Haemophilus influenzae is a ribonuclease. J Bacteriol 2007, 189:5041-5048.

30. Salzano AM, Febbraio F, Farias T, Cetrangolo GP, Nucci R, Scaloni A, Manco G: Redox stress proteins are involved in adaptation response of the hyperthermoacidophilic archaeon Sulfolobus solfataricus to nickel challenge. Microb Cell Fact 2007, 6:25.

31. Schelert J, Drozda M, Dixit V, Dillman A, Blum P: Regulation of mercury resistance in the crenarchaeote Sulfolobus solfataricus. J Bacteriol 2006, 188:7141-7150. ●● Discusses the use of genetic tools in S. solfataricus to elucidate the regulation of a mercury resistance mechanism.

32. Remonsellez F, Orell A, Jerez CA: Copper tolerance of the thermoacidophilic archaeon Sulfolobus metallicus: possible role of polyphosphate metabolism. Microbiology 2006, 152:59-66. ● A study of Cu efflux and correlation to polyP accumulation and enzyme activity in selected Sulfolobus species with high and low Cu tolerances.

33. Ettema TJ, Brinkman AB, Lamers PP, Kornet NG, de Vos WM, van der Oost J: Molecular characterization of a conserved archaeal copper resistance (cop) gene cluster and its copper-responsive regulator in Sulfolobus solfataricus P2. Microbiology 2006, 152:1969-1979.

34. Kim S, Lee SB: Rare codon clusters at 5'-end influence heterologous expression of archaeal gene in Escherichia coli. Protein Expr Purif 2006, 50:49-57.

35. Koma D, Sawai T, Harayama S, Kino K: Overexpression of the genes from thermophiles in Escherichia coli by high-temperature cultivation. Appl Microbiol Biotechnol 2006, 73:172-180.

36. Albers S-V, Driessen AJ: Conditions for gene disruption by homologus recombination of exogenous DNA into the Sulfolobus solfataricus genome. Archaea 2007, 2:145-149.

37. Worthington P, Hoang V, Perez-Pomares F, Blum P: Targeted disruption of the alpha-amylase gene in the hyperthermophilic archaeon Sulfolobus solfataricus. J Bacteriol 2003, 185:482-488.

38. Martin A, Yeats S, Janekovic D, Reiter WD, Aicher W, Zillig W: SAV 1, a temperate u.v.-inducible DNA virus-like particle from the archaebacterium Sulfolobus acidocaldarius isolate B12. EMBO J 1984, 3:2165-2168.

39. Clore AJ, Stedman KM: The SSV1 viral integrase is not essential. Virology 2007, 361:103-111.

40. Aucelli T, Contursi P, Girfoglio M, Rossi M, Cannio R: A spreadable, non-integrative and high copy number shuttle vector for Sulfolobus solfataricus based on the genetic element pSSVx from Sulfolobus islandicus. Nucleic Acids Res 2006, 34:e114.

41. Berkner S, Grogan D, Albers SV, Lipps G: Small multicopy, non-integrative shuttle vectors based on the plasmid pRN1 for Sulfolobus acidocaldarius and Sulfolobus solfataricus, model organisms of the (cren-)archaea. Nucleic Acids Res 2007, 35:e88.

42. Albers SV, Jonuscheit M, Dinkelaker S, Urich T, Kletzin A, Tampe R, Driessen AJ, Schleper C: Production of recombinant and tagged proteins in the hyperthermophilic archaeon Sulfolobus solfataricus. Appl Environ Microbiol 2006, 72:102-111.

43. Schelert J, Dixit V, Hoang V, Simbahan J, Drozda M, Blum P: Occurrence and characterization of mercury resistance in the hyperthermophilic archaeon Sulfolobus solfataricus by use of gene disruption. J Bacteriol 2004, 186:427-437.

44. Du Plessis CA, Batty JD, Dew DW: Commercial Applications of Thermophile Bioleaching. In Biomining. Edited by Rawlings DE, Johnson DB: Springer-Verlag; 2007.

45. Mikkelsen D, Kappler U, McEwan AG, Sly LI: Archaeal diversity in two thermophilic chalcopyrite bioleaching reactors. Environ Microbiol 2006, 8:2050-2056.

46. Rawlings DE, Johnson DB: The microbiology of biomining: development and optimization of mineral-oxidizing microbial consortia. Microbiology 2007, 153:315- 324. ● Consortia optimization strategy considerations based on microbiology of biocatalysts, as well as chemical engineering characteristics of biomining operations.

47. Chen L, Brugger K, Skovgaard M, Redder P, She Q, Torarinsson E, Greve B, Awayez M, Zibat A, Klenk HP, et al.: The genome of Sulfolobus acidocaldarius, a model organism of the Crenarchaeota. J Bacteriol 2005, 187:4992-4999.

48. Kawarabayasi Y, Hino Y, Horikawa H, Jin-no K, Takahashi M, Sekine M, Baba S, Ankai A, Kosugi H, Hosoyama A, et al.: Complete genome sequence of an aerobic thermoacidophilic crenarchaeon, Sulfolobus tokodaii strain7. DNA Res 2001, 8:123-140.

49. She Q, Singh RK, Confalonieri F, Zivanovic Y, Allard G, Awayez MJ, Chan-Weiher CC- Y, Clausen Ib G, Curtis BA, De Moors A, et al.: The complete genome of the crenarchaeon Sulfolobus solfataricus P2. Proceedings of the National Academy of Sciences of the United States of America 2001, 98:7835-7840.

50. Auernik KS, Maezato Y, Blum PH, Kelly RM: The genome sequence of the metal- mobilizing, extremely thermoacidophilic archaeon Metallosphaera sedula provides insights into bioleaching-associated metabolism. Appl Environ Microbiol 2008, 74:682-692. ● Discusses genome features relevant to biomining and genome-wide transcriptional response to the presence of ferrous iron.

51. Castresana J, Lubben M, Saraste M: New archaebacterial genes coding for redox proteins: implications for the evolution of aerobic metabolism. J Mol Biol 1995, 250:202-210.

52. Lubben M, Arnaud S, Castresana J, Warne A, Albracht SP, Saraste M: A second terminal oxidase in Sulfolobus acidocaldarius. Eur J Biochem 1994, 224:151-159.

53. Purschke WG, Schmidt CL, Petersen A, Schafer G: The terminal quinol oxidase of the hyperthermophilic archaeon Acidianus ambivalens exhibits a novel subunit structure and gene organization. J Bacteriol 1997, 179:1344-1353.

54. Hiller A, Henninger T, Schafer G, Schmidt CL: New genes encoding subunits of a cytochrome bc1-analogous complex in the respiratory chain of the hyperthermoacidophilic crenarchaeon Sulfolobus acidocaldarius. J Bioenerg Biomembr 2003, 35:121-131.

55. Kappler U, Sly LI, McEwan AG: Respiratory gene clusters of Metallosphaera sedula - differential expression and transcriptional organization. Microbiology 2005, 151:35-43.

56. Bathe S, Norris PR: Ferrous iron- and sulfur-induced genes in Sulfolobus metallicus. Appl Environ Microbiol 2007, 73:2491-2497. ●● Identification of a new terminal oxidase cluster and ETC components involved in ferrous iron oxidation in selected Sulfolobus species.

57. Urich T, Gomes CM, Kletzin A, Frazao C: X-ray Structure of a self- compartmentalizing sulfur cycle metalloenzyme. Science 2006, 311:996-1000. ● Structural resolution of an intracellular sulfur oxidoreductase and implications for its mechanism of operation via creation of a microenvironment.

58. Kletzin A: Metabolism of inorganic sulfur compounds in Archaea. In Archaea: Evolution, Physiology, and Molecular Biology. Edited by Garrett RA, Klenk HP: Blackwell; 2007:261-276. ● A thorough summary of known inorganic sulfur metabolism in archaea and comparison to bacterial metabolism.

59. Berg IA, Kockelkorn D, Buckel W, Fuchs G: A 3-hydroxypropionate/4- hydroxybutyrate autotrophic carbon dioxide assimilation pathway in Archaea. Science 2007, 318:1782-1786. ●● Outline of a new inorganic carbon fixation cycle in archaea and phylogenetic diversity of microorganisms containing key enzymes.

60. Hugler M, Krieger RS, Jahn M, Fuchs G: Characterization of acetyl-CoA/propionyl- CoA carboxylase in Metallosphaera sedula. Carboxylating enzyme in the 3- hydroxypropionate cycle for autotrophic carbon fixation. Eur J Biochem 2003, 270:736-744.

61. Alber B, Olinger M, Rieder A, Kockelkorn D, Jobst B, Hugler M, Fuchs G: Malonyl- coenzyme A reductase in the modified 3-hydroxypropionate cycle for autotrophic carbon fixation in archaeal Metallosphaera and Sulfolobus spp. J Bacteriol 2006, 188:8551-8559.

62. Alber BE, Kung JW, Fuchs G: 3-Hydroxypropionyl-coenzyme A synthetase from Metallosphaera sedula, an enzyme involved in autotrophic CO2 fixation. J Bacteriol 2008, 190:1383-1389.

63. Tang TH, Polacek N, Zywicki M, Huber H, Brugger K, Garrett R, Bachellerie JP, Huttenhofer A: Identification of novel non-coding RNAs as potential antisense regulators in the archaeon Sulfolobus solfataricus. Mol Microbiol 2005, 55:469-481.

64. Omer AD, Zago M, Chang A, Dennis PP: Probing the structure and function of an archaeal C/D-box methylation guide sRNA. RNA 2006, 12:1708-1720.

65. Ciammaruconi A, Gorini S, Londei P: A bifunctional archaeal protein that is a component of 30S ribosomal subunits and interacts with C/D box small RNAs. Archaea 2007, 2:151-158.

66. Lillestol RK, Redder P, Garrett RA, Brugger K: A putative viral defence mechanism in archaeal cells. Archaea 2006, 2:59-72.

67. Sorek R, Kunin V, Hugenholtz P: CRISPR - a widespread system that provides acquired resistance against phages in bacteria and archaea. Nat Rev Microbiol 2007.

68. Sleytr UB, Egelseer EM, Ilk N, Pum D, Schuster B: S-Layers as a basic building block in a molecular construction kit. FEBS J 2007, 274:323-334.

69. Engelhardt H: Are S-layers exoskeletons? The basic function of protein surface layers revisited. J Struct Biol 2007, 160:115-124.

70. Engelhardt H: Mechanism of osmoprotection by archaeal S-layers: a theoretical study. J Struct Biol 2007, 160:190-199.

71. Bode ML, Buddoo SR, Minnaar SH, du Plessis CA: Extraction, isolation and NMR data of the tetraether lipid calditoglycerocaldarchaeol (GDNT) from Sulfolobus metallicus harvested from a bioleaching reactor. Chem Phys Lipids 2008.

72. Huber H, Stetter KO: Desulfurococcales. In The Prokaryotes, edn 3. Edited by Dworkin M, Falkow S, Rosenberg E, Schleifer K, Stackebrandt E: Springer; 2006:52-68.

73. Huber H, Huber R, Stetter KO: Thermoproteales. In The Prokaryotes, edn 3. Edited by Dworkin M, Falkow S, Rosenberg E, Schleifer K, Stackebrandt E: Springer; 2006:10-22.

Table 1.1: Selected features of extreme thermoacidophiles with sequenced genomes. Compiled from NCBI May 2008 genome project links (http://www.ncbi.nlm.nih.gov/) and references listed for this article.

Genome Estimated Sequenced Growth Growth Sequence GC # of Thermoacidophile Temp Growth Optimum reported content Genome encoded IS toxin-like hypothetical (oC) pH (oC, pH) Isolated from (%) Size (Mb) proteins elements1 COGs2 proteins Crenarchaea-Thermoprotei

Thermoproteales: 85,

Caldivirga maquilingensis 60-92 2.3-6.4 3.7-4.2 acidic hot spring, Philippines 2007 43.1 2.1 1963 7 15 549

Sulfolobales:

solfataric thermal pond drainage, Metallosphaera sedula 50-80 1-4.5 75, 2-3 Italy 2007 46.2 2.2 2256 11 18 673

70-75,

Sulfolobus acidocaldarius 55-85 1-6 2-3 solfataric hot spring, Italy 2005 36.7 2.2 2223 14 19 1002

Sulfolobus solfataricus 50-87 2-5.5 85, 3-4.5 solfataric hot spring, Italy 2001 35.8 3 2977 159 26 1340

Sulfolobus tokodaii 70-85 2-5.5 80, 2.5-3 hot spring, Japan 2001 32.8 2.7 2825 12 32 1874

(Table 1.1 continued)

Euryarchaea-Thermoplasmata

Thermoplasmatales

Picrophilus torridus 47-65 0-3.5 60, 0.7 volcanic solfataric field, Japan 2004 36 1.5 1535 4 4 291

Thermoplasma acidophilum 45-63 0.5-4 59, 1-2 burning coal refuse pile, USA 2001 46 1.6 1482 4 6 583

Thermoplasma volcanium 33-67 1-4 60, 2 volcanic solfataric field, Italy 2001 39.9 1.6 1499 56 6 261

1annotation contains "transposase", "integrase", or "resolvase" 2ORFs similar to COGs 1412, 1439, 1487, 1848, 3413, or 4113 containing nucleic acid binding and/or PIN (PilT N terminus) domains

Figure 1.1: Unrooted 16S phylogenentic tree (constructed using ClustalW and Phylip 3.5c o available on http://mobyle.pasteur.fr) of extremely thermoacidophilic archaea (Topt ≥60 C, S pHopt <4), compiled from [8,9,11,14,72,73]. Genomes are denoted as sequenced or Sipsequencing in progress, according to www.genomesonline.org. Organisms denoted in “ ” have not yet been described in full detail. Accession numbers for 16S rRNA are listed in parentheses. Sso = S. solfataricus (SSOr03), “Sis”=”S. islandicus” strain M14A (AY247895), Ssh=S. shibatae (M32504), Sto=S. tokodaii (ABO22438), “Syang”= ”Sulfolobus yangmingensis” (AB010957), Sowh= Sulfurisphaera ohwakuensis (D85507), Saci= S. acidocaldarius (D14876), “Sthur” = “Sulfolobus thuringensis” (X90485), Sazo=Stygiolobus azoricus (D85520), Smet= S. metallicus (U40813), Msed=M. sedula (Msed_R0026), Mpru= Metallosphaera prunae (X90482), Mhak=Metallosphaera hakonensis (D86414), Abr=Acidianus brierleyi (X90477), “Aten”= ”Acidianus tengchongensis” (AF226987), Ainf=A. infernus (X89852), Aam=Acidianus ambivalens (D85506), Asuf=Acidianus sulfidivorans (AY907891), Aac=Acidolobus aceticus (AF191225), Cmaq=Caldivirga maquilingensis (ABO13926), Posh=Picrophilus oshimae (X84901), Pto=Picrophilus torridus (PTOr02), Taci=Thermogymnomonas acidicola (AB269873), Tvn=Thermoplasma volcanium (Tvnr04), DHVE2 represented by 16S of A. boonei (DQ451875). No 16S sequences are available for extreme thermoacidophiles Sulfurococcus yellowsonensis and Sulfurococcus mirabilis.

Figure 1.2: Metal resistance mechanisms in extreme thermoacidophiles. CopA is the P- type ATPase shown to be involved in copper and cadmium cation efflux in S. solfataricus [33]. MerA is the mercuric reductase which reduces soluble Hg2+ to volatile elemental Hg and is constitutively expressed in S. solfataricus [31]. Ppk (polyphosphate kinase) and Ppx (exopolyphosphatase) which comprise the polyP system described in S. metallicus [32].

Figure 1.3: The recently proposed 5th cycle of autotrophic carbon fixation, adapted from[59]. 1. acetyl-CoA/propionyl-CoA carboxylase, 2. malonyl-CoA/succinyl-CoA reductase, 3. malonate semialdehyde reductase, 4. 3-hydroxypropionyl-CoA synthetase, 5. 3-hydroxypropionyl-CoA dehydratase, 6. acryloyl-CoA reductase 7. methylmalonyl-CoA epimerase, 8. metylmalonyl-CoA mutase, 9. succinate semialdehyde reductase, 10. 4- hydroxybutyryl-CoA synthetase, 11. 4-hydroxybutyryl-CoA dehydratase, 12. crotonyl-CoA hydratase, 13. 3-hydroxybutaryl-CoA dehydrogenase, 14. acetoacetyl-CoA β-keothiolase. ORFs encoding enzymes with (?) have not been finalized in the extreme thermoacidophile in which the cycle was studied. Many of the intermediates of this cycle are the same as found in the 3-hydroxypropionate cycle (left side and base of triangle). The right side of triangle represents rearrangement after the second carbon addition, concluding with a split into two molecules of acetyl-CoA, which distinguishes the 3-hydroxpropionate/4- hydroxybutyrate cycle from the 3-hydroxypropionate cycle.

28 Chapter 2

The genome sequence of the metal-mobilizing, extremely thermoacidophilic archaeon Metallosphaera sedula provides insights into bioleaching-associated metabolism

Kathryne S. Auernik, Yukari Maezato#, Paul H. Blum#, and Robert M. Kelly

Department of Chemical and Biomolecular Engineering North Carolina State University Raleigh, NC 27695-7905

#Beadle Center for Genetics University of Nebraska-Lincoln Lincoln, NE 68588-0666

Published in Applied and Environmental Microbiology 74(3): 682-692 (2008)

29 ABSTRACT

Despite their taxonomic description, not all Sulfolobales are capable of oxidizing reduced sulfur species, which along with iron oxidation, make up two desirable traits of biomining microorganisms. However, the complete genome sequence of the extremely thermoacidophilic archaeon Metallosphaera sedula DSM 5348 (2.2 Mb, ~2300 ORFs) provides insights into biologically catalyzed metal sulfide oxidation. Comparative genomics was used to identify pathways and proteins (in)directly involved with bioleaching. As expected, the M. sedula genome encodes genes related to autotrophic carbon fixation, metal tolerance, and adhesion. Also, terminal oxidase cluster organization indicates the presence of hybrid quinol-cytochrome oxidase complexes. Comparisons with the mesophilic biomining bacterium Acidithiobacillus ferrooxidans ATCC 23270 indicate that the

M. sedula genome encodes at least one putative rusticyanin, involved in iron oxidation, and a putative tetrathionate hydrolase, implicated in sulfur oxidation. The fox gene cluster, involved in iron oxidation in the thermoacidophilic archaeon Sulfolobus metallicus, could also be identified. These iron- and sulfur-oxidizing components are missing from genomes of non-leaching Sulfolobales like Sulfolobus solfataricus P2 and Sulfolobus acidocaldarius

DSM 639. Whole genome transcriptional response analysis showed that 88 ORFs were up- regulated 2-fold or more in M. sedula upon addition of ferrous sulfate to yeast extract-based medium; these included components of terminal oxidase clusters predicted to be involved with iron oxidation, as well as genes predicted to be involved with sulfur metabolism. Many hypothetical proteins were also differentially transcribed, indicating that aspects of the iron and sulfur metabolism of M. sedula remain to be identified and characterized.

30 INTRODUCTION

Biomining exploits acidophilic microorganisms to recover valuable metals (i.e., Cu,

Au) from ores (52, 56, 57, 59) in biohydrometallurgical processes conducted at temperatures ranging from ambient (44, 71) to 80°C (17). Higher temperature operations involve consortia of extremely thermoacidophilic archaea from the genera Sulfolobus,

Acidianus, and Metallosphaera (49, 50). For example, Sulfolobus metallicus, certain

Acidianus species (12, 47), and Metallosphaera sedula (29, 33) all can mobilize metals from metal sulfides. However, not all members of these genera are metal bioleachers. In fact, the three Sulfolobus species with completed genome sequences (S. solfataricus, S. acidocaldarius, and S. tokodai) are from microorganisms apparently unable to effect metal sulfide oxidation. Since genome sequences are not yet available for metal bioleaching extreme thermoacidophiles, genetic features characteristic of this physiological capability remain to be seen.

Biomining is based upon biological oxidation of iron (Fe) and reduced inorganic sulfur compounds (RISCs). Two Fe(III)-driven chemical leaching mechanisms are catalyzed by microorganisms regenerating Fe(III) from Fe(II), facilitating release of valuable metals bound in metal sulfides (52, 56, 57, 59). As accumulation of precipitating RISC compounds can slow leaching rates (52), microbial RISC-oxidizing capabilities are beneficial. Extreme thermophiles are particularly desirable, as problematic passivation on ore surfaces decreases at higher temperatures. Since chemolithotrophs use Fe(II) and/or RISCs as energy sources, nutritional supplement costs are relatively low, thus not impacting process economics.

31 Components of electron transport chains involved in Fe and RISC oxidation are differentially expressed as a function of substrate in both meso- and thermoacidophilic bioleachers (5, 36, 55). Bacterial Fe oxidation studies focused on mesophilic A. ferrooxidans have shown that electrons from Fe(II) can either be transported along a

“downhill” or “uphill” electron pathway, towards a lower (O2/H2O) or higher

(NAD(P)+/NAD(P)H) potential redox couple, respectively (28). Both pathways involve electron transfer via cytochromes c and rusticyanin (Rus), a periplasmic blue-copper protein

(Bcp),. The downhill and uphill pathways, encoded by the rus and petI operons, conclude with reduction of the final target by a cytochrome c oxidase (CoxABCD) and bc1 complex

(PetI), respectively, and are highly transcribed on Fe(II) relative to elemental sulfur (So) (7,

18, 72). In a proposed archaeal Fe oxidation model, based on Ferroplasma strains, the terminal oxidase combines select bc1-complex-like and cytochrome c oxidase-like components and involves association of a Bcp, sulfocyanin (15). Aspects of this model were noted previously in the non-leacher S. acidocaldarius (39, 41), while a correlation between cytochromes b, b558/562 (CbsAB), and Fe-S cluster proteins (possible quinol oxidase components) and the bc1 complex has been proposed (27, 39, 65, 75). Finally, a gene cluster was identified in S. metallicus, containing terminal oxidase and electron transport chain components, highly transcribed on Fe(II) vs So (5).

Extremely thermoacidophilic archaeal RISC oxidation mechanisms have not been established. RISC electrons may enter the electron transport chain via a membrane-bound

2- thiosulfate (S2O3 ):quinone oxidoreductase (TQO), a sulfite:acceptor oxidoreductase

(SAOR), or possibly through a sulfide:quinone oxidoreductase (SQR) (38). Despite the

32 annotation, tetrathionate hydrolases (TetH) have been noted in A. ferrooxidans and

o 2- Acidithiobacillus caldus expressed on S , FeS2, and S2O3 substrates (8, 35, 61).

Certain terminal oxidase components have been expressed in extremely thermoacidophilic archaea when grown on specific RISCs (36, 54). Although not directly linked to electron transport, sulfur oxygenase reductases (Sor) are believed to be important for breakdown of intracellular RISCs, particularly So or polysulfides (11, 37, 69). M. sedula

o growth on S and metal sulfides (i.e. FeS2 and chalcopyrite (CuFeS2)), has been

2- documented (29). With S2O3 as a primary intermediate of acid-insoluble metal sulfide leaching, this may be a RISC substrate. However, there are no reports of M. sedula growth

2- 2- on S2O3 or other RISCs, such as sulfite (SO3 ) or polythionates.

Although M. sedula presumably grows mixotrophically, autotrophy is critical in biomining environments, where relatively small amounts of organic carbon are available.

Several Sulfolobales, including M. sedula, purportedly fix carbon (CO2) via a modified 3- hydroxypropionate cycle, identified in Chloroflexus aurantiacus (31-34, 43, 67). In M. sedula, two enzymes of this cycle have been characterized biochemically (1, 33), while two additional enzymes’ activities were detected in autotrophically-grown cell extracts, suggesting their involvement in CO2 fixation (32).

Heavy metals, typically toxic in concentrations higher than trance amounts, are routinely encountered by biomining acidophiles. In contrast to substantial information about metal toxicity in neutrophiles (48), relatively little is known about such mechanisms in acidophiles, despite their higher resistance levels (16). Toxicity and mechanisms of resistance to mercury (Hg) have been investigated in S. solfataricus (14, 63, 64), while possible copper (Cu)-resistance mechanisms have been studied in S. solfataricus and S.

33 metallicus (19, 58). Continued study of heavy metal toxicity and resistance mechanisms in bioleachers is important, especially for cases like M. sedula, where tolerance to some

metals, particularly Cu, may require improvement to make it competitive with other microbial biominers, such as S. metallicus and A. ferrooxidans (16, 30, 58).

Adhesion mechanisms of biomining microorganisms to acid-insoluble metal sulfides or So may be important to oxidation rates. Study of bioleachers’ extracellular polymeric substances, often polysaccharides (EPS), has centered on A. ferrooxidans (6, 13, 23, 24,

51, 62). EPS production, composition, and properties were all observed to be a function of growth substrate in A. ferrooxidans (23, 24). In addition, significant homology is suggested between the components of the bacterial Type II secretion, Type IV pilus, and archaeal flagellar (Fla) systems (53). Bacterial type IV pili and Fla systems have been implicated in adhesion (46, 66), and pilus-like structures, along with “wiggling of the cells at the ore”, were observed during initial characterization of M. sedula (29).

To better understand the defining features of extreme thermoacidophiles capable of metal sulfide oxidation, the genome sequence of M. sedula was completed and probed for clues with respect to five physiological areas (iron oxidation, sulfur oxidation, carbon fixation, metal toxicity, and adhesion) that are important to its current and future role in biomining operations. In addition, whole genome transcriptional response analysis was used to identify specific ORFs related to Fe oxidation.

34 MATERIALS AND METHODS

Sequencing of the M. sedula genome. M. sedula (DSM 5348T) was purified to clonality by aerobic cultivation at 70°C on a solid medium, prepared as described previously

(64), and adjusted to a pH of 2.5 with sulfuric acid. High molecular weight genomic DNA was isolated, as described previously (26), and provided to the US Department of Energy

Joint Genome Institute (http://www.jgi.doe.gov/) for cloning and shotgun sequencing. A combination of small (average insert sizes: 3, 8 kb) and large (40 kb, fosmid) insert libraries were prepared and used for analysis as indicated at http://www.jgi.doe.gov/.

Nucleotide sequence accession number. The 18 contigs of the draft genome sequence were made available publicly on 27-Nov-2006, with the final assembly released on 25-Apr-2007 and listed under GenBank accession #CP000682 (http://genome.jgi- psf.org/finished_microbes/metse/metse.info.html).

Annotation. Critica, Generation, and Glimmer software programs were used for coding region detection and gene identification. TMMHMM 2.0

(http://www.cbs.dtu.dk/services/TMHMM/) was used to predict transmembrane helices in translated sequences. SignalP v2.0b2 (http://www.cbs.dtu.dk/services/SignalP-2.0/) was used to predict the presence and location of N-terminal signal peptides.

Comparative analysis. For comparative genomics, M. sedula gene sequences and annotations were downloaded from the JGI microbial genome website

35 (http://genome. ornl.gov/microbial/msed/28feb07/). Other sequences referenced were obtained from GenBank, with the exception of A. ferrooxidans ATCC 23270 sequences, which were obtained from The Institute for Genome Research’s Comprehensive Microbial

Resource (TIGR CMR). The Basic Local Alignment Search Tool (BLAST) searches were conducted using a BLOSUM62 matrix with either the NCBI protein-protein blast program

(BLASTP) against the Swissprot or nr databases, or the ORNL Microbial BLAST server’s

BLASTP program against the msed database. “Significant similarity” or “significant homology” was defined as ≥ 20% identity and Eval ≤ 10-5, unless otherwise noted.

Electron microscopy. Samples used for scanning electron microscopy were

o grown aerobically at 70 C in a minimal salts medium (60) containing 0.1% FeS2 as sole energy source; prior to use, the FeS2 was sterilized by baking for three days at 140°C.

Glutaraldehyde fixed cells treated with osmium tetroxide were applied to poly-L- lysine coated, gold-palladium treated coverslips, followed by ethanol dehydration. Following critical point drying, gold-palladium coated cells were visualized using a Hitachi S-3000N variable pressure scanning electron microscope (http://bsweb.unl.edu/labs/blum/index.html).

Transcriptional response analysis. An oligonucleotide microarray was developed using a minimum of one 60-mer probe (designed using OligoArray 2.1 software, synthesized by IDT) for each identifiable protein-encoding ORF in the M. sedula genome.

Cells (DSMZ 5348) were grown aerobically at 70oC in an orbital shaking oil bath at 70 rpm on DSMZ 88 medium (pH 2), supplemented with 0.1% yeast extract (YE). An initial density of mid 106 cells/mL was used in four 1 L bottles containing 300 mL volume of the same

36 medium. Approximately 7.5 g/L of FeSO4·7H2O was added to two of these cultures prior to inoculation. At harvest, cultures in mid-exponential phase (~24 h of growth, mid-107 cells/mL) were quickly chilled and then centrifuged at 9510 x g for 15 minutes at 4oC. RNA was extracted and purified (RNAqueous, Ambion), reverse transcribed (Superscript III,

Invitrogen), re-purified, labeled with either Cy3 or Cy5 dye (GE Healthcare), and hybridized to one of two microarray slides (Corning). Slides were scanned on a Perkin-Elmer scanner and raw intensities were quantitated using ScanArray® Express 2.1 software.

Normalization of data and statistical analysis were performed using JMP Genomics 6.0.2 software (SAS Institute, Cary, NC).

RESULTS AND DISCUSSION

Organization and general features of the genome. The M. sedula genome (46%

G+C) encodes 2258 proteins, 35% of which are annotated as either “hypothetical protein” or “protein of unknown function”. However, almost 90% of these have their strongest similarity (“top hit”) to Sulfolobus species (786 to S. solfataricus, 829 to S. tokodaii, 327 to

S. acidocaldarius, 14 to S. islandicus, 3 to S. shibatae), with 35 open reading frames

(ORFs) most similar to genes in Acidianus species. This raises some interesting questions.

Will the genes responsible for the biomining capabilities of M. sedula be found within the

10% of gene sequences with strongest similarities to non-sulfolobales or non-archaeal microorganisms? Or, conversely, will subtle differences in the sulfolobales’ gene sequences result in the distinction between thermoacidophilic leachers and non-leachers?

37 Iron oxidation. The Fe oxidation mechanism in M. sedula is, as yet, unknown. The organization and composition of at least 5 respiratory clusters found in the genome sequence, two of which are predicted to be highly transcribed during growth on Fe(II) or

FeS2, differs from the bacterial rus and pet operons. No PetC-like ORFs (cytochrome c subunit of the bc1 complex) are apparent. This is consistent with reports that archaeal genomes of Ferroplasma and Sulfolobus species do not contain cytochromes c (68) and suggests that acidophilic archaeal and bacterial Fe oxidation do not occur via the same mechanism. The gene neighborhood organization of respiratory clusters in M. sedula provides support for the Ferroplasma hybrid terminal oxidase model.

Four Bcp-like proteins are found in the genome; two are annotated as hypothetical sulfocyanins (SoxE). Figure 2.1 displays a multiple sequence alignment of these Bcps, along with similar Bcp-like sequences from other microorganisms. Almost all the Bcp sequences appear to contain four ligands important for Cu-binding and the characteristically high positive redox potential of Rus in A. ferrooxidans (4, 76). Both Msed0323 and

Msed0826 contain two recognized protein signatures for sulfocyanins, while the two remaining Bcps from M. sedula (Msed0966 and 1206) contain rusticyanin protein signatures (25). The SoxE-like sequences appear to align fairly well with each other, but the same cannot be said for the Rus-like sequences. The relevance of poor alignment between

Rus-like sequences, as it relates to protein function, is not clear. The archaeal Rus-like proteins cannot have the same cellular location as their bacterial periplasmic counterpart.

Msed0966 is more similar to the Rus found in A. ferrooxidans (Afe3186) than Msed1206.

Both have predicted N-terminal signal peptides, but Msed1206 also contains a single, predicted C-terminal hydrophobic transmembrane region. Although the other sequenced

38 Sulfolobus species contain multiple SoxE homologs, only S. solfataricus (Sso1870) appears to contain a Rus-like (Msed0966-like) sequence. However, Sso1870 does not appear to contain a recognized Rus protein signature or the four ligands noted as important to Cu- binding and redox potential.

Three sets of ORFs with low similarity to the PetAB subunits of the bc1 complex are found in the M. sedula genome (Msed0288 and 0291, Msed0322-1, and Msed0501-0)

(Figure 2.2). In each case, a Rieske protein (SoxL/SoxF) pairs with a cytochrome b

(SoxC/SoxG/SoxN). In the first two cases, the Rieske and Cyt b are clustered with cytochrome oxidase (Cox) subunits I and II (SoxAB or SoxHM) at the same loci, suggesting close association between quinol- and Cox-like subunits. In the case of Locus 3, there is another CoxI-II pair located further downstream (Locus 4) which lacks a corresponding

Rieske-Cyt b (PetAB-like) pair. However, it is not clear if loci 3 and 4 should be grouped into a single, more diffuse, locus. Both loci contain a cytochrome b558/562 (CbsAB, Msed 0503-4 and Msed0477-8). Limited information available makes it difficult to determine whether

CbsAB could substitute for quinol oxidase or Cox components of a terminal oxidase, however previous studies have suggested CbsAB may play a role in extracellular electron transport similar to the bc1 complex (27, 65, 75). Locus 5 encodes DoxBCE (Msed2030-2), the SoxAB-like terminal oxidase components from A. ambivalens, and appears to be missing quinol oxidase-like subunits. Interestingly, Msed1191 appears to encode a lone quinol oxidase fusion protein (cytochrome b N-terminal and Rieske C-terminal).

Because Locus 1 soxB (Msed0290) was reported to be up-regulated on So, compared to FeS2 or YE (36), and DoxBCE expression and co-purification with TQO subunits (DoxDA) was noted under So-oxidizing conditions in A. ambivalens (45, 54), it

39 seems likely that the Loci 1 and 5 respiratory clusters in M. sedula would also be stimulated during growth on RISCs. Locus 2 soxM (Msed0324) was reported as up-regulated on YE,

o compared to FeS2 or S (36). Locus 3 cbsA (Msed0504) was found to have higher

o transcription on FeS2 than YE or S (36); therefore the entire locus is predicted to be up- regulated on Fe(II). Interestingly, Locus 3 follows a proton efflux ATPase (Msed0505).

Previous A. ferrooxidans studies suggested this type of ATPase could function in either direction to regulate a cytochrome oxidase on the basis of available ATP, restoring proton motive force (PMF) or supporting NADP(H) reduction (18). Locus 4 contains a gene cluster with homology to fox genes in S. tokodaii and S. metallicus, which were demonstrated to be up-regulated on Fe(II) vs So (5). While Locus 3 components appear to be present in both S. acidocaldarius and S. solfataricus genomes (27, 65), neither genome contains fox homologs (excepting FoxHJ in Sso and FoxH in Sac). This is not entirely surprising, as neither organism appears capable of growth on Fe(II). In light of this observation, the fox gene cluster is likely essential for metal mobilization. Because S. tokodaii was recently reported to grow on Fe(II) (5) and its genome does not seem to encode Rus, it is not clear whether rusticyanin is essential for archaeal Fe oxidation or if an alternative redox protein, such as sulfocyanin (SoxE), may provide an equivalent function. It is interesting to note that

Locus 4 includes a Cox II homolog (soxH, Msed0480) which contains conserved Cu-binding ligands similar to Locus 2 SoxH (Msed0320) of the SoxM supercomplex thought to interact with sulfocyanin (Msed0323), also of the SoxM supercomplex (39). Taken together, the predicted Fe oxidation capacity encoded in Locus 4 could involve sulfocyanin, rusticyanin, or both. Because Msed1191 is unique to the BLAST database (as of Sept 2007), no similar

40 sequence could be identified to predict what type of substrate could induce transcription of this ORF.

Sulfur oxidation. M. sedula encodes a pair of proteins (Msed0363-4) with significant similarity to DoxDA subunits of the TQO in A. ambivalens (45). At least two

SAOR-like proteins exist in M. sedula: a putative sulfite oxidase (Msed0362) on the strand opposite doxDA and an A. ferrooxidans CysI-like (Afe3210) putative sulfite reductase

(Msed0961) near a putative rusticyanin; however, neither appears to be membrane- associated. Of the 5 ORFs in M. sedula (Msed0353, 0558, 1039, 1323, 2059) with significant similarity to putative SQR proteins in A. ferrooxidans (Afe1293, 2761), only

Msed1039 is predicted to contain a signal peptide. The M. sedula genome encodes a membrane-associated protein (Msed0804) with significant similarity to TetH found in both

A. ferrooxidans (Afe2996) and Acidithiobacillus caldus (ABP38225). No counterparts can be identified in either S. solfataricus or S. acidocaldarius, although Sto1164 appears to be similar to the TetH of A. ferrooxidans. Neither an archaeal nor a bacterial Sor sequence is evident in the genome of M. sedula. While Sor is also apparently absent from the genomes of S. solfataricus and S. acidocaldarius, it is present in S. tokodaii and F. acidarmanus.

Putative versions of adenosine phosphosulfate (APS) reductase and sulfate adenylyl transferase (SAT) are co-located in M. sedula (Msed0962-3), on the strand opposite a putative sulfite reductase (Msed0961). Depending on operational direction, these proteins could link RISCs to substrate level phosphorylation. Operating oxidatively, as

2- seen in chemolithotrophs, APS “reductase” converts SO3 to APS, followed by SAT oxidation of APS to sulfate and ATP (74). Operating reductively, as seen in heterotrophs

41 and autotrophs, requires function of SAT and then APS reductase, converting sulfate to

2- 2- SO3 . A sulfite reductase further reduces SO3 to sulfide and then cysteine is formed via an o-acetyl-L-serine(thiol) lyase (70) or a cysteine synthase. Similar to M. sedula, the A. ferrrooxidans 23270 genome also encodes a collocated APS-SAT pair, although the SAT sequence does not appear to be similar to that of M. sedula. SAT is usually encoded by 2 genes, an ATP sulfurylase and an APS kinase, although there are instances of their fusion

(74). However, in M. sedula, only the ATP sulfurylase component is found, suggesting that

APS, and not phosphoadenosine phosphosulfate (PAPS), is formed. This is supported by the APS reductase sequence, which contains 4 cysteines (for 4Fe-4S center coordination) characteristic of APS reductase but missing in PAPS reductase (70). Similarly, the CysI-like sulfite oxidoreductase subunit (Msed0961) is found in A. ferrooxidans along with a flavoprotein subunit (CysJ) (70); however, in M. sedula, the CysJ-like component appears to be absent. Because the APS-SAT pair appears in non-leaching Sulfolobus species, the importance of these proteins in a bioleaching microorganism is unclear.

Finally, one putative rusticyanin mentioned previously (Msed0966) is located on the strand opposite of the APS-SAT pair in M. sedula. The clustering of this gene relative to those involved with sulfur metabolism may be coincidental; however, it may also suggest a physiological link between Fe and RISC oxidation important for bioleaching. Preliminary support for this hypothesis is based on an A. ferrooxidans study, which showed rusticyanin to be up-regulated during early exponential growth phase on a sulfur substrate (55).

Carbon metabolism. Sequences of the two previously characterized enzymes of the 3-hydroxypropionate cycle (catalyzing steps 1, 2, and 4) can be located in the genome

42 (see Figure 2.3). Potential sequences for previously detected enzymatic activities of propionyl-CoA synthase (step 3) and fumarate hydratase (step 9) activities were also found.

Although no propionyl-CoA synthase is annotated in the M. sedula genome, there are multiple AMP-dependent synthetase/ligase candidates, with Msed1353 having the most sequence similarity to the N-terminal propionyl-CoA synthases from both C. aurantiacus

(AAL47820) and Roseiflexus RS-1 (YP_001277513) (20). It is interesting to note that both bacterial enzymes are 2-3 times the size of Msed1353 and raises the prospect that a multi- subunit propionyl-CoA synthase complex exists in M. sedula. Querying amino acids ~900-

1100 and ~1300-1500 of both bacterial enzymes against the M. sedula genome results in hits to ORFs automatically annotated as enoyl-CoA hydratases (or 3-hydroxybutaryl-CoA dehydratases) and alcohol dehydrogenases, respectively. Two classes of fumarate dehydratase were also identified (Msed1115-6, class I; Msed1462, class II). Candidates for the six additional enzymatic steps (5,6,8,10, 11, and 14) of the cycle can be identified in the genome sequence, with the predicted assignment of Msed0381 to steps 10, 11, and 14 based on its sequence similarity to the single enzyme in C. aurantiacus (mcl) responsible for malyl-, β-methylmalyl-, and S-citramalyl-CoA lyase activity (20, 42). Recent information suggests that at least part of the pathways in C. aurantiacus and M. sedula evolved independently (1). Hence, it is not surprising to find that the genome is devoid of sequences with similarity to encoded enzymes responsible for transferase activity in C. aurantiacus

(21, 22) (steps 7 and 10). Because components of this pathway identified in M. sedula are not clustered, genome location is of limited use in determining remaining enzymes participating in the pathway.

43 Metal tolerance. Microbial P-type ATPases, specifically CPX-ATPase, have been proposed to mediate efflux of heavy metal cations, such as Cu, Zn, and Cd (16, 48, 58). A

CPX-ATPase has been identified in M. sedula (Msed0490), with a C-P-C pattern located in the 5th of 7 predicted transmembrane regions. This gene has significant homology to a P- type ATPase in S. solfataricus (copA) implicated in Cu(II), and possibly also cadmium, cation efflux (19). Ettema et al. (19) demonstrated that the metal efflux process involved a metallochaperone (CopM), having a 32bp overlap with CopA, and a new type of archaeal transcriptional regulator (CopT). M. sedula contains ORFs with significant similarity to both

CopM and CopT (Msed0491-2). However, the copMA overlap is limited to 10bp, and the location of copT (on the opposite strand) makes the gene organization reminiscent of versions encoded in the S. tokodaii and S. acidocaldarius genomes.

With respect to the polyphosphate mechanism proposed for Cu(II) detoxification in

S. metallicus (58), Msed0740 is annotated as a probable polyphosphate/NAD kinase

(ppnk), and Msed0981 has 37% similarity to the Ppx previously characterized in S. solfataricus (9). Searching for possible metal-phosphate complex exporters, no significant hits to E. coli phosphate transport systems (pstSCAB or Pit) were identified in M. sedula.

However, as found with other genome-sequenced acidophiles (2, 58), use of a S. cervisiae

Pho84 sequence query produced results: 3 hits with similarities of 25-30% (Msed1512,

Msed1094, Msed0866). Using the top Pho84-like sequence in A. ferrooxidans (Afe0861) as a template, 4 hits with similarities of 30-32% resulted (previous 3 + Msed0846). All 4 hits belong to the major facilitator superfamily of substrate transporters. Msed1521 encodes a hypothetical protein downstream of Msed1512 with weak similarity a phosphate transport

44 regulation protein (COG1392, PFAM01865). Msed1968 (COG0704) is a putative phosphate uptake regulator.

A gene corresponding to an arsenite transporter (ArsB) was also identified

(Msed2086, COG1055). The As(III) membrane potential-driven pump is typically found along with a repressor protein (ArsR) and sometimes with an As(V) reductase (ArsC), an As resistance boosting APTase (ArsA), and/or a secondary regulation protein (ArsD)(16). In M. sedula, however, ArsB appears to stand alone. Msed1005 potentially encodes an ArsR-like regulatory protein with a TRASH domain, but it is unclear whether this protein might be involved in the regulation of ArsB, MerAH (Msed1241-2), or some other metal resistance mechanism.

Adhesion. Figure 2.4 depicts M. sedula attached to FeS2 and apparent EPS production. In support of this observation, the Msed1805-1857 locus encodes ORFs potentially involved in EPS production. Queries from A. ferrooxidans (Afe1738) and V. cholerae (vps21) produced hits with significant similarity to Msed1810. All three sequences are annotated as a UDP-glucose (or mannose) 6-dehydrogenase (EC 1.1.22). Msed1809, which shares a 3bp overlap with Msed1810, has lower, but significant, similarity to Afe1739, although both sequences are annotated as UDP glucose 4-epimerase. Msed1808, annotated as a hypothetical protein, shares significant similarity with glycosyltransferases of

S. auereus (capM) and V. cholerae (vps32) implicated in capsular polysaccharide biosynthesis (40, 73). Msed1811, annotated as a UDP-glucose pyrophosphorylase (EC

2.7.7.9), is not related to the EPS query sequences used, but like Msed1808-10, may encode enzymes involved in the synthesis of biofilm components, specifically glucuronic

45 acid via the Leloir pathway (3). EPS of FeSO4- and FeS2-grown cells of A. ferrooxidans contain Fe(III) complexed with glucuronic acid, which is believed to impart an overall positive charge to the EPS, promoting attachment to FeS2, which tends to maintain an overall negative charge at low pH (62). Additional ORFs located further downstream in this

1800s region have annotations suggestive of a role in formation of other EPS components

(i.e. UDP-galactose, dTDP-rhamnose), but specific biochemical functions and cellular roles of the encoded proteins remain to be seen.

Genome analysis reveals at least 4 gene clusters with similarities to the Type

II/IV/Fla systems. Msed1324-1330 appears to encode 7 flagellar proteins. Msed1324, annotated as FlaJ, contains 8 predicted transmembrane regions and shares a 14bp overlap with Msed1325. Msed1325-6 (FlaIH) encodes 2 cytoplasmic subunits related to ATPases

(COG0630, COG2874), while Msed1327-9 overlap with each other, by 1bp (Msed1327-8) or 23bp (Msed1328-9), and are all predicted to have 1 N-terminal transmembrane helix.

Msed1327-8 corresponds to FlaFG, while Msed1329 shares no identity with characterized proteins. Msed1330 is annotated as a flagellin protein and has similarity to the major preflagellin/prepilin FlaB genes (COG1681). Outside of this cluster, there are at least 3 other pairs of FlaIJ-like proteins (Msed1197-8, Msed2104-5, Msed0650-1). In the first two cases, FlaI-like genes are annotated as Type II secretion system proteins, while their overlapping FlaJ-like genes are annotated as hypothetical proteins. In the last case, however, both genes are annotated as hypotheticals and share no overlap. Although all 3

FlaI-like proteins are closely related to Msed1325, none of the 3 FlaJ-like proteins are similar to Msed1324. Msed1198 has significant similarity to Msed2105, but neither are similar to Msed0651. Also of note is a prepilin/preflagellin peptidase located upstream of

46 one of these FlaIJ pairs (Msed2090). With 237aa and 6 predicted transmembrane helices, this FlaK-like sequence fits the characteristic size range and α-helix predictions for bacterial

Type II/IV systems (53).

Transcriptional response of M. sedula to FeSO4. To assess whether specific aspects of bioinformatics analysis of M. sedula genome sequence have a physiological basis, transcriptional profiling using a whole genome oligonucleotide microarray was pursued. The addition of FeSO4 to a YE-supplemented media triggered differential transcription of 88 genes (≥2.0 fold), or ~4% of the genome (see Figure 2.5 and Table

2.A1). Of these, 53 were more highly transcribed in the presence of FeSO4, including several M. sedula ORFs predicted to be involved with Fe oxidation, sulfur metabolism, and adhesion. Six ORFs, most with only a general “major facilitator superfamily” annotation, were up-regulated in the presence of FeSO4+YE compared to YE (Msed0467, 0907, 1001,

1095, 1181, and 1355). Table 2.1 lists differentially transcribed ORFs predicted to be involved with Fe oxidation, sulfur metabolism, and adhesion, while Figure 2.6 summarizes sequence-based predictions and preliminary transcriptome implications for these same three physiological functions. With respect to Fe oxidation, a putative rusticyanin

(Msed1206), the Locus 3 respiratory cluster (Msed0500-4) expected to be up-regulated on

Fe(II) and a unique putative quinol oxidase fusion protein (cytochrome b N-terminal and

Rieske C-terminal domain) were highly transcribed in the presence of FeSO4. Several fox genes of Locus 4 (specifically Msed0477-0480) were also highly transcribed on FeSO4, although levels in the presence of YE alone were at the upper end of the dynamic response range of the array. The exception to this was subunit I of the terminal oxidase (SoxB,

47 Msed0484), which was more highly transcribed in the presence of FeSO4+YE. The implied involvement of M. sedula Locus 4 genes in Fe oxidation builds on previous reports on the closely-related autotrophic biomining microorganism, S. metallicus (5, 30), and the influence of a heterotrophic substrate (YE) compared to FeSO4 has not previously been investigated. ORFs potentially involved in sulfur metabolism (Msed0960-3,

CysGIHN-like sequences, respectively) were up-regulated in the presence of

FeSO4. With the addition of a sulfur compound in the +6 valence state, these ORFs, including the APS-SAT pair, may be functioning in the reductive direction, reducing sulfate to sulfide. It is interesting that a cysteine synthase (CysM)-like ORF (Msed1607) was down- regulated on FeSO4+YE. This may be an attempt to limit accumulation of cysteine, which could lead to Fenton reaction oxidative damage to DNA (70), although it is not immediately apparent how excess sulfide is alternatively processed. Additionally, Msed1197-8, similar to cytoplasmic ATPase and transmembrane subunits, were stimulated in the presence of

FeSO4 compared to YE alone. This FlaIJ pair was one of 4 Fla clusters predicted to be involved with adhesion, either through flagella formation or EPS secretion. Although the 3 remaining Fla clusters may not function as predicted, it is also possible that their transcription has yet to be induced by the appropriate substrate. EPS production, composition, and properties were observed to be a function of substrate in A. ferrooxidans,

(13, 24, 62). Finally, nearly half of the differentially transcribed ORFs in the YE/YE+FeSO4 contrast were “hypothetical proteins” or “proteins of unknown function”, indicating that novel proteins are involved in Fe oxidation metabolism in M. sedula. Efforts to understand Fe oxidation and other elements of M. sedula physiology using functional genomics approaches are underway.

Summary. Comparing genome sequences of extremely thermoacidophilic non- leachers, mesoacidophilic bioleachers, and M. sedula provides a number of preliminary insights into the genotype that supports high temperature biomining. Such comparisons between M. sedula and genome-sequenced Sulfolobales indicate that the fox gene cluster, two putative versions of Rus, and TetH appear to be factors distinguishing thermoacidophilic bioleachers from non-bioleachers. However, it is still not clear whether bioleachers have acquired these genes or non-leachers have lost them (or their functionality). For example, the genome of the apparent non-leacher, S. solfataricus, encodes a protein similar to a putatative Rus in M. sedula, but is lacking a Rus protein signature and apparent Cu-binding ligands. S. tokodaii was recently demonstrated to have some degree of Fe oxidation capability, and contains several fox genes(5), as well as a

TetH, but is lacking a Rus-like sequence. Finally, the terminal oxidase components (Locus

3) shown to be up-regulated on Fe(II) here, as well as another study(36), were originally described in the apparent non-leacher, S. acidocaldarius (27). The differential transcription of significant numbers of hypothetical proteins in YE+FeSO4(vs. YE)-grown M. sedula cells confirms there are many other proteins involved in Fe oxidation and/or sulfur metabolism which may help to further distinguish between bioleachers and non-leachers in the future.

The future success of the biomining industry will depend upon the performance of cellular biocatalysts. The availability of the M. sedula genome sequence has advanced both functional genomics and genetics efforts in this area. To understand performance, upcoming functional genomics studies will focus on M. sedula oxidation of RISCs and

49 reduced Fe substrates, and how these processes relate to cell adhesion to solid inorganic substrates. Functional genomics analyses can also help guide genetic tools in target selection. Genetic tools in development for M. sedula (Y. Maezato and P. Blum, unpublished) will be important for examining and enhancing performance of pathways and metal-microbe interactions essential to biomining operations at elevated temperatures.

ACKNOWLEDGMENTS

The authors gratefully acknowledge the US Department of Energy and the Joint

Genome Institute for providing the genome sequence for Metallosphaera sedula. This work was supported in part by a grant from the U.S. National Science Foundation Biotechnology

Program (No. BES-0317886). KSA acknowledges support from an NIH T32 Biotechnology

Traineeship.

REFERENCES

1. Alber, B. E., M. Olinger, A. Rieder, D. Kockelkorn, B. Jobst, M. Hugler, and G. Fuchs. 2006. Malonyl-CoA reductase in the modified 3-hydroxypropionate cycle for autotrophic carbon fixation in archaeal Metallosphaera and Sulfolobus sp. J. Bacteriol. 188:8551-9.

2. Alvarez, S., and C. A. Jerez. 2004. Copper ions stimulate polyphosphate degradation and phosphate efflux in Acidithiobacillus ferrooxidans. Appl. Environ. Microbiol. 70:5177-82.

3. Barreto, M., E. Jedlicki, and D. S. Holmes. 2005. Identification of a gene cluster for the formation of extracellular polysaccharide precursors in the

50 chemolithoautotroph Acidithiobacillus ferrooxidans. Appl. Environ. Microbiol. 71:2902-9.

4. Barrett, M. L., I. Harvey, M. Sundararajan, R. Surendran, J. F. Hall, M. J. Ellis, M. A. Hough, R. W. Strange, I. H. Hillier, and S. S. Hasnain. 2006. Atomic resolution crystal structures, EXAFS, and quantum chemical studies of rusticyanin and its two mutants provide insight into its unusual properties. Biochemistry 45:2927-39.

5. Bathe, S., and P. R. Norris. 2007. Ferrous iron- and sulfur-induced genes in Sulfolobus metallicus. Appl. Environ. Microbiol. 73:2491-7.

6. Bevilaqua, D., I. Diez-Perez, C. S. Fugivara, F. Sanz, A. V. Benedetti, and O. Garcia, Jr. 2004. Oxidative dissolution of chalcopyrite by Acidithiobacillus ferrooxidans analyzed by electrochemical impedance spectroscopy and atomic force microscopy. Bioelectrochemistry 64:79-84.

7. Bruscella, P., C. Appia-Ayme, G. Levican, J. Ratouchniak, E. Jedlicki, D. S. Holmes, and V. Bonnefoy. 2007. Differential expression of two bc1 complexes in the strict acidophilic chemolithoautotrophic bacterium Acidithiobacillus ferrooxidans suggests a model for their respective roles in iron or sulfur oxidation. Microbiology 153:102-10.

8. Buonfiglio, V., M. Polidoro, F. Soyer, P. Valenti, and J. Shively. 1999. A novel gene encoding a sulfur-regulated outer membrane protein in Thiobacillus ferrooxidans. J. Biotechnol. 72:85-93.

9. Cardona, S. T., F. P. Chavez, and C. A. Jerez. 2002. The exopolyphosphatase gene from sulfolobus solfataricus: characterization of the first gene found to be involved in polyphosphate metabolism in archaea. Appl. Environ. Microbiol. 68:4812-9.

10. Castresana, J., M. Lubben, and M. Saraste. 1995. New archaebacterial genes coding for redox proteins: implications for the evolution of aerobic metabolism. J. Mol. Biol. 250:202-10.

11. Chen, Z. W., Y. Y. Liu, J. F. Wu, Q. She, C. Y. Jiang, and S. J. Liu. 2007. Novel bacterial sulfur oxygenase reductases from bioreactors treating gold-bearing concentrates. Appl. Microbiol. Biotechnol. 74:688-98.

51 12. Clark, T. R., F. Baldi, and G. J. Olson. 1993. Coal Depyritization by the Thermophilic Archaeon Metallosphaera sedula. Appl. Environ. Microbiol. 59:2375- 2379.

13. Devasia, P., K. A. Natarajan, D. N. Sathyanarayana, and G. R. Rao. 1993. Surface Chemistry of Thiobacillus ferrooxidans Relevant to Adhesion on Mineral Surfaces. Appl. Environ. Microbiol. 59:4051-4055.

14. Dixit, V., E. Bini, M. Drozda, and P. Blum. 2004. Mercury inactivates transcription and the generalized transcription factor TFB in the archaeon Sulfolobus solfataricus. Antimicrob. Agents Chemother. 48:1993-9.

15. Dopson, M., C. Baker-Austin, and P. L. Bond. 2005. Analysis of differential protein expression during growth states of Ferroplasma strains and insights into electron transport for iron oxidation. Microbiology 151:4127-37.

16. Dopson, M., C. Baker-Austin, P. R. Koppineedi, and P. L. Bond. 2003. Growth in sulfidic mineral environments: metal resistance mechanisms in acidophilic microorganisms. Microbiology 149:1959-70.

17. Du Plessis, C. A., J. D. Batty, and D. W. Dew. 2007. Commercial Applications of Thermophile Bioleaching. In D. E. Rawlings and D. B. Johnson (ed.), Biomining. Springer-Verlag, Berlin Heidelberg.

18. Elbehti, A., G. Brasseur, and D. Lemesle-Meunier. 2000. First evidence for existence of an uphill electron transfer through the bc(1) and NADH-Q oxidoreductase complexes of the acidophilic obligate chemolithotrophic ferrous ion- oxidizing bacterium Thiobacillus ferrooxidans. J. Bacteriol. 182:3602-6.

19. Ettema, T. J., A. B. Brinkman, P. P. Lamers, N. G. Kornet, W. M. de Vos, and J. van der Oost. 2006. Molecular characterization of a conserved archaeal copper resistance (cop) gene cluster and its copper-responsive regulator in Sulfolobus solfataricus P2. Microbiology 152:1969-79.

20. Friedmann, S., B. E. Alber, and G. Fuchs. 2007. Properties of R-citramalyl- coenzyme A lyase and its role in the autotrophic 3-hydroxypropionate cycle of Chloroflexus aurantiacus. J. Bacteriol. 189:2906-14.

52 21. Friedmann, S., B. E. Alber, and G. Fuchs. 2006. Properties of succinyl-coenzyme A:D-citramalate coenzyme A transferase and its role in the autotrophic 3- hydroxypropionate cycle of Chloroflexus aurantiacus. J. Bacteriol. 188:6460-8.

22. Friedmann, S., A. Steindorf, B. E. Alber, and G. Fuchs. 2006. Properties of succinyl-coenzyme A:L-malate coenzyme A transferase and its role in the autotrophic 3-hydroxypropionate cycle of Chloroflexus aurantiacus. J. Bacteriol. 188:2646-55.

23. Gehrke, T., R. Hallmann, K. Kinzler, and W. Sand. 2001. The EPS of Acidithiobacillus ferrooxidans--a model for structure-function relationships of attached bacteria and their physiology. Water Sci. Technol. 43:159-67.

24. Gehrke, T., J. Telegdi, D. Thierry, and W. Sand. 1998. Importance of Extracellular Polymeric Substances from Thiobacillus ferrooxidans for Bioleaching. Appl. Environ. Microbiol. 64:2743-7.

25. Giri, A. V., S. Anishetty, and P. Gautam. 2004. Functionally specified protein signatures distinctive for each of the different blue copper proteins. BMC Bioinformatics 5:127.

26. Haseltine, C., R. Montalvo-Rodriguez, E. Bini, A. Carl, and P. Blum. 1999. Coordinate transcriptional control in the hyperthermophilic archaeon Sulfolobus solfataricus. J. Bacteriol. 181:3920-7.

27. Hiller, A., T. Henninger, G. Schafer, and C. L. Schmidt. 2003. New genes encoding subunits of a cytochrome bc1-analogous complex in the respiratory chain of the hyperthermoacidophilic crenarchaeon Sulfolobus acidocaldarius. J. Bioenerg. Biomembr. 35:121-31.

28. Holmes, D. S., and V. Bonnefoy. 2007. Genetic and Bioinformatic Insights into Iron and Sulfur Oxidation Mechanisms of Bioleaching Organisms. In D. E. Rawlings and D. B. Johnson (ed.), Biomining. Springer-Verlag, Berlin Heidelberg.

29. Huber, G., C. Spinnler, A. Gambacorta, and K. O. Stetter. 1989. Metallosphaera sedula gen. and sp. nov. represents a new genus of aerobic, metal-mobilizing, thermoacidophilic archaebacteria. Syst. Appl. Microbiol. 12:38-47.

53 30. Huber, G., and K. O. Stetter. 1991. Sulfolobus metallicus, sp. nov., a novel strictly chemolithoautotrophic thermophilic archaeal species of metal-mobilizers. Syst. Appl. Microbiol. 14:372-378.

31. Hugler, M., and G. Fuchs. 2005. Assaying for the 3-hydroxypropionate cycle of carbon fixation. Methods Enzymol. 397:212-21.

32. Hugler, M., H. Huber, K. O. Stetter, and G. Fuchs. 2003. Autotrophic CO2 fixation pathways in archaea (Crenarchaeota). Arch. Microbiol. 179:160-73.

33. Hugler, M., R. S. Krieger, M. Jahn, and G. Fuchs. 2003. Characterization of acetyl-CoA/propionyl-CoA carboxylase in Metallosphaera sedula. Carboxylating enzyme in the 3-hydroxypropionate cycle for autotrophic carbon fixation. Eur. J. Biochem. 270:736-44.

34. Ishii, M., S. Chuakrut, H. Arai, and Y. Igarashi. 2004. Occurrence, biochemistry and possible biotechnological application of the 3-hydroxypropionate cycle. Appl. Microbiol. Biotechnol. 64:605-10.

35. Kanao, T., K. Kamimura, and T. Sugio. 2007. Identification of a gene encoding a tetrathionate hydrolase in Acidithiobacillus ferrooxidans. J. Biotechnol. 132:16-22.

36. Kappler, U., L. I. Sly, and A. G. McEwan. 2005. Respiratory gene clusters of Metallosphaera sedula - differential expression and transcriptional organization. Microbiology 151:35-43.

37. Kletzin, A. 1989. Coupled enzymatic production of sulfite, thiosulfate, and hydrogen sulfide from sulfur: purification and properties of a sulfur oxygenase reductase from the facultatively anaerobic archaebacterium Desulfurolobus ambivalens. J. Bacteriol. 171:1638-43.

38. Kletzin, A., T. Urich, F. Muller, T. M. Bandeiras, and C. M. Gomes. 2004. Dissimilatory oxidation and reduction of elemental sulfur in thermophilic archaea. J. Bioenerg. Biomembr. 36:77-91.

39. Komorowski, L., W. Verheyen, and G. Schafer. 2002. The archaeal respiratory supercomplex SoxM from S. acidocaldarius combines features of quinole and cytochrome c oxidases. Biol. Chem. 383:1791-9.

54 40. Lin, W. S., T. Cunneen, and C. Y. Lee. 1994. Sequence analysis and molecular characterization of genes required for the biosynthesis of type 1 capsular polysaccharide in Staphylococcus aureus. J. Bacteriol. 176:7005-16.

41. Lubben, M., S. Arnaud, J. Castresana, A. Warne, S. P. Albracht, and M. Saraste. 1994. A second terminal oxidase in Sulfolobus acidocaldarius. Eur. J. Biochem. 224:151-9.

42. Meister, M., S. Saum, B. E. Alber, and G. Fuchs. 2005. L-malyl-coenzyme A/beta- methylmalyl-coenzyme A lyase is involved in acetate assimilation of the isocitrate lyase-negative bacterium Rhodobacter capsulatus. J. Bacteriol. 187:1415-25.

43. Menendez, C., Z. Bauer, H. Huber, N. Gad'on, K. O. Stetter, and G. Fuchs. 1999. Presence of acetyl coenzyme A (CoA) carboxylase and propionyl-CoA carboxylase in autotrophic Crenarchaeota and indication for operation of a 3-hydroxypropionate cycle in autotrophic carbon fixation. J. Bacteriol. 181:1088-98.

44. Morin, D. H. R., and P. D'Hugues. 2007. Bioleaching of a Colbalt-Containing Pyrite in Stirred Reactors: a Case Study from Laboratory Scale to Industrial Application. In D. E. Rawlings and D. B. Johnson (ed.), Biomining. Springer-Verlag, Berlin Heidelberg.

45. Muller, F. H., T. M. Bandeiras, T. Urich, M. Teixeira, C. M. Gomes, and A. Kletzin. 2004. Coupling of the pathway of sulphur oxidation to dioxygen reduction: characterization of a novel membrane-bound thiosulphate:quinone oxidoreductase. Mol. Microbiol. 53:1147-60.

46. Nather, D. J., R. Rachel, G. Wanner, and R. Wirth. 2006. Flagella of Pyrococcus furiosus: multifunctional organelles, made for swimming, adhesion to various surfaces, and cell-cell contacts. J. Bacteriol. 188:6915-23.

47. Nemati, M., J. Lowenadler, and S. T. Harrison. 2000. Particle size effects in bioleaching of pyrite by acidophilic thermophile Sulfolobus metallicus (BC). Appl. Microbiol. Biotechnol. 53:173-9.

48. Nies, D. H. 2003. Efflux-mediated heavy metal resistance in prokaryotes. FEMS Microbiol. Rev. 27:313-39.

55 49. Norris, P. R. 2007. Acidophile Diversity in Mineral Sulfide Oxidation. In D. E. Rawlings and D. B. Johnson (ed.), Biomining. Springer-Verlag, Berlin Heidelberg.

50. Norris, P. R., N. P. Burton, and N. A. Foulis. 2000. Acidophiles in bioreactor mineral processing. Extremophiles 4:71-6.

51. Ohmura, N., Kitamura, Keiko; Saiki, Hiroshi. 1993. Selective Adhesion of Thiobacillus ferrooxidans to Pyrite. Appl. Environ. Microbiol. 59:4044-4050.

52. Olson, G. J., J. A. Brierley, and C. L. Brierley. 2003. Bioleaching review part B: progress in bioleaching: applications of microbial processes by the minerals industries. Appl. Microbiol. Biotechnol. 63:249-57.

53. Peabody, C. R., Y. J. Chung, M. R. Yen, D. Vidal-Ingigliardi, A. P. Pugsley, and M. H. Saier, Jr. 2003. Type II protein secretion and its relationship to bacterial type IV pili and archaeal flagella. Microbiology 149:3051-72.

54. Purschke, W. G., C. L. Schmidt, A. Petersen, and G. Schafer. 1997. The terminal quinol oxidase of the hyperthermophilic archaeon Acidianus ambivalens exhibits a novel subunit structure and gene organization. J. Bacteriol. 179:1344-53.

55. Ramirez, P., N. Guiliani, L. Valenzuela, S. Beard, and C. A. Jerez. 2004. Differential protein expression during growth of Acidithiobacillus ferrooxidans on ferrous iron, sulfur compounds, or metal sulfides. Appl. Environ. Microbiol. 70:4491- 8.

56. Rawlings, D. E. 2005. Characteristics and adaptability of iron- and sulfur-oxidizing microorganisms used for the recovery of metals from minerals and their concentrates. Microb. Cell Fact. 4:13.

57. Rawlings, D. E., D. Dew, and C. du Plessis. 2003. Biomineralization of metal- containing ores and concentrates. Trends Biotechnol. 21:38-44.

58. Remonsellez, F., A. Orell, and C. A. Jerez. 2006. Copper tolerance of the thermoacidophilic archaeon Sulfolobus metallicus: possible role of polyphosphate metabolism. Microbiology 152:59-66.

56 59. Rohwerder, T., T. Gehrke, K. Kinzler, and W. Sand. 2003. Bioleaching review part A: progress in bioleaching: fundamentals and mechanisms of bacterial metal sulfide oxidation. Appl. Microbiol. Biotechnol. 63:239-48.

60. Rolfsmeier, M., and P. Blum. 1995. Purification and characterization of a maltase from the extremely thermophilic crenarchaeote Sulfolobus solfataricus. J. Bacteriol. 177:482-5.

61. Rzhepishevska, O. I., J. Valdes, L. Marcinkeviciene, C. A. Gallardo, R. Meskys, V. Bonnefoy, D. S. Holmes, and M. Dopson. 2007. Regulation of a Novel Acidithiobacillus caldus Gene Cluster Involved in Reduced Inorganic Sulfur Compound Metabolism. Appl. Environ. Microbiol. 73:7367-72.

62. Sand, W., and T. Gehrke. 2006. Extracellular polymeric substances mediate bioleaching/biocorrosion via interfacial processes involving iron(III) ions and acidophilic bacteria. Res. Microbiol. 157:49-56.

63. Schelert, J., V. Dixit, V. Hoang, J. Simbahan, M. Drozda, and P. Blum. 2004. Occurrence and characterization of mercury resistance in the hyperthermophilic archaeon Sulfolobus solfataricus by use of gene disruption. J. Bacteriol. 186:427- 37.

64. Schelert, J., M. Drozda, V. Dixit, A. Dillman, and P. Blum. 2006. Regulation of mercury resistance in the crenarchaeote Sulfolobus solfataricus. J. Bacteriol. 188:7141-50.

65. Schoepp-Cothenet, B., M. Schutz, F. Baymann, M. Brugna, W. Nitschke, H. Myllykallio, and C. Schmidt. 2001. The membrane-extrinsic domain of cytochrome b(558/566) from the archaeon Sulfolobus acidocaldarius performs pivoting movements with respect to the membrane surface. FEBS Lett. 487:372-6.

66. Skerker, J. M., and H. C. Berg. 2001. Direct observation of extension and retraction of type IV pili. Proc. Natl. Acad. Sci. U. S. A. 98:6901-4.

67. Strauss, G., and G. Fuchs. 1993. Enzymes of a novel autotrophic CO2 fixation pathway in the phototrophic bacterium Chloroflexus aurantiacus, the 3- hydroxypropionate cycle. Eur. J. Biochem. 215:633-43.

57 68. Tyson, G. W., J. Chapman, P. Hugenholtz, E. E. Allen, R. J. Ram, P. M. Richardson, V. V. Solovyev, E. M. Rubin, D. S. Rokhsar, and J. F. Banfield. 2004. Community structure and metabolism through reconstruction of microbial genomes from the environment. Nature 428:37-43.

69. Urich, T., C. M. Gomes, A. Kletzin, and C. Frazao. 2006. X-ray Structure of a self- compartmentalizing sulfur cycle metalloenzyme. Science 311:996-1000.

70. Valdes, J., F. Veloso, E. Jedlicki, and D. Holmes. 2003. Metabolic reconstruction of sulfur assimilation in the extremophile Acidithiobacillus ferrooxidans based on genome analysis. BMC Genomics 4:51.

71. van Aswegen, P. C., van Niekerk, J., Olivier, W. 2007. The BIOX(TM) Process for the Treatment of Refractory Gold Concentrates. In D. E. Rawlings and D. B. Johnson (ed.), Biomining. Springer-Verlag, Berlin Heidelberg.

72. Yarzabal, A., C. Appia-Ayme, J. Ratouchniak, and V. Bonnefoy. 2004. Regulation of the expression of the Acidithiobacillus ferrooxidans rus operon encoding two cytochromes c, a cytochrome oxidase and rusticyanin. Microbiology 150:2113-23.

73. Yildiz, F. H., and G. K. Schoolnik. 1999. Vibrio cholerae O1 El Tor: identification of a gene cluster required for the rugose colony type, exopolysaccharide production, chlorine resistance, and biofilm formation. Proc. Natl. Acad. Sci. U. S. A. 96:4028- 33.

74. Yu, Z., E. B. Lansdon, I. H. Segel, and A. J. Fisher. 2007. Crystal structure of the bifunctional ATP sulfurylase-APS kinase from the chemolithotrophic thermophile Aquifex aeolicus. J. Mol. Biol. 365:732-43.

75. Zahringer, U., H. Moll, T. Hettmann, Y. A. Knirel, and G. Schafer. 2000. Cytochrome b558/566 from the archaeon Sulfolobus acidocaldarius has a unique Asn-linked highly branched hexasaccharide chain containing 6-sulfoquinovose. Eur. J. Biochem. 267:4144-9.

76. Zeng, J., M. Geng, Y. Liu, L. Xia, J. Liu, and G. Qiu. 2007. The sulfhydryl group of Cys138 of rusticyanin from Acidithiobacillus ferrooxidans is crucial for copper binding. Biochim. Biophys. Acta 1774:519-25.

58 Table 2.1: Transcriptional response of YE-grown M. sedula to ferrous sulfate. For reference, significance values of 1.3, 4.3, and 9.3 correlate to p-values of 0.05, 5 x 10-5, and 5 x 10-10, respectively.

Bioleaching Msed GenBank Annotation fold change: Significancea Annotation characteristic ORF# YE+ FeSO4 vs YE -log10 (p-value) 0477 Hypothetical 1.8 ↓ 2.6 Putative cbsB/foxD 0478 Hypothetical 3.3 ↓ 7.3 Putative cbsA/foxC 0479 Hypothetical 5.4 ↓ 8.0 hypothetical protein Iron oxidation 0480 Cytochrome c oxidase (subunit II) 3.8 ↓ 10.5 Putative cytochrome oxidase (soxH/foxB) (Locus 4) 0484 Cytochrome c oxidase (subunit I) 1.8 ↑ 4.5 Putative cytochrome oxidase (soxB/foxA) 0500 Cytochrome b/b6, N-terminal domain 3.0 ↑ 4.1 Putative quinol oxidase (soxN) 0501 Rieske (2Fe-2S) domain 1.9 ↑ 3.0 Putative quinol oxidase (soxL) Iron oxidation 0503 Hypothetical 1.7 ↑ 3.6 Putative cbsB (Locus 3) 0504 Hypothetical 2.0 ↑ 1.7 Putative cbsA Sulfur 0960 Uroporphyrin-III C/tetrapyrrole (Corrin/Porphyrin) Methyltransferase 2.5 ↑ 9.3 Putative uroporphyrin III C-methyltransferase metabolism 0961 Nitrite and sulphite reductase Putative sulfite reductase 4Fe-4S region 2.9 ↑ 8.5 0962 Adenylylsulfate reductase, thioredoxin dependent 3.2 ↑ 5.9 APS reductase 0963 Sulfate adenylyltransferase 1.9 ↑ 3.1 Sulfate adenylyltransferase Iron oxidation 1191 Rieske (2Fe-2S) domain 4.3 ↑ 4.0 Putative quinol oxidase fusion (soxN-soxL) Adhesion 1197 Type II secretion system protein E 12.4 ↑ 11.4 Putative ATPase (flaI) 1198 Type II secretion system protein 6.0 ↑ 6.3 Putative flaJ Iron oxidation 1206 Blue (type 1) copper domain 1.8 ↑ 4.4 Putative rusticyanin

Figure 2.1: Selected sections of a full multiple sequence alignment of known and putative proteins involved in Fe oxidation performed by CLUSTAL W (1.83). Amino acids known or predicted to be involved with copper binding (4, 10, 25, 76) are shown in bold. Segments underlined and highlighted in gray and black show protein signatures for sulfocyanin and rusticyanin, respectively, with intermediate non-highlighted amino acids failing to meet currently recognized positional consensus amino acids (25). Organism name and gene annotation are abbreviated, with sso = S. solfataricus, sac = S. acidocaldarius, sto = S. tokodaii, msed = M. sedula, faci = F. acidarmanus, , afe = A. ferrooxidans, soxE = sulfocyanin, hyp = hypothetical protein, bcp = blue copper protein, rus = rusticyanin.

Figure 2.2: Proposed organization of common elements in terminal oxidase complexes of M. sedula. Terminal oxidase complexes have been proposed to be hybrids of bacterial bc1 complexes, containing FeS Rieske (”SoxL” or “SoxF”) and cytochrome b (“SoxC”, “SoxG”, or “SoxN”) components, and cytochrome oxidases, containing Cox subunits I and II (“SoxB” or “SoxM” and “SoxA” or “SoxH”, respectively). Also provided is the relative genome sequence location (ORF numbers) of these common elements; previous studies of these general subunit or cluster functions are listed at the far right. Sme = S. metallicus; Aam = A. ambivalens.

Figure 2.3: The 3-hydroxypropionate cycle that is thought to operate in C. aurantiacus, adapted from (20, 21). ORF numbers predicted to encode the required enzymes are specified. 1) acetyl-CoA carboxylase, 2) malonyl-CoA reductase, 3) propionyl-CoA synthase, 4) propionyl-CoA carboxylase, 5) methylmalonyl-CoA epimerase, 6) methylmalonyl-CoA mutase, 7) succinyl-CoA:L-malyl-CoA transferase, 8) succinate dehydrogenase, 9) fumarate dehydratase, 10) L-malyl-CoA lyase, 11) β-methylmalyl-CoA lyase, 12) unknown enzymes, 13) succinyl-CoA: citramalate-CoA transferase, 14) S/R- citramalyl-CoA lyase

Figure 2.4: M. sedula attached to a FeS2 substrate. Bar = 2 μm.

Figure 2.5: Volcano plot of M. sedula microarray dye-flip results (YE vs. YE+FeSO4). Negative x-axis values represent ORFs more highly transcribed on YE+FeSO4 while positive x-axis values represent ORFs more highly transcribed on YE alone. As points of reference on the y-axis, p-values of 0.05, 5 x 10-5, and 5 x 10-10 would correspond to significance values of 1.3, 4.3, and 9.3, respectively.

Figure 2.6: Schematic summarizing Fe oxidation, sulfur metabolism, and adhesion components believed to be important to M. sedula’s biomining capacity. Elements of the schematic are linked to their annotations or putative functions by superscript numbers. Both genome sequence analysis and preliminary transcriptional profiling results (FeSO4+YE vs. YE) indicate that membrane-associated proteins have a prevalent role in biomining physiology.

64 Chapter 2 Appendix

The genome sequence of the metal-mobilizing, extremely thermoacidophilic archaeon Metallosphaera sedula provides insights into bioleaching-associated metabolism

Table 2.A1: All ORFs differentially expressed 2.0-fold (log2 fold=1.0) or higher.

-log10(p- value) for Diff Lsmean trt Lsmean trt Log2 fold change of trt = (YE)- Gene ID YE YEF = (YE)-(YEF) (YEF) GenBank Annotation (19Aug2007) Manual Annotation Notes/Comments

Msed0012 0.768845 -0.19164 0.960484 2.557062 Prephenate dehydratase

Msed0028 1.595192 0.626336 0.968857 3.098885 ribosomal protein L24E Msed0062 1.68294 0.603746 1.079194 4.478858 RNA polymerase Rpb4

Msed0149 -1.82099 1.675228 -3.49622 13.31622 exonuclease of the beta-lactamase fold involved in RNA processing-like protein hypothetical exonuclease

Msed0161 1.334999 0.379099 0.955899 2.650253 hypothetical protein Msed_0161 hypothetical protein

Msed0203 0.669646 -0.44877 1.118416 4.364733 ribosomal protein L40e 50S ribosomal protein L40e

Msed0236 2.268856 0.986089 1.282767 4.55907 sugar isomerase (SIS) hypothetical D-arabino-3-hexulose-6-phosphate isomerase

Msed0242 0.307795 1.410793 -1.103 7.341228 peptidase M48, Ste24p hypothetical Zn-dependent protease

Msed0267 0.251303 1.372984 -1.12168 1.685441 AMP-dependent synthetase and ligase putative acetyl-CoA synthetase

Msed0307 3.130844 4.136216 -1.00537 5.510023 pyruvate ferredoxin/flavodoxin oxidoreductase, delta subunit Msed0327 -1.20552 0.139928 -1.34545 4.73837 hypothetical protein Msed_0327

Msed0328 2.922364 1.76271 1.159654 3.837116 hypothetical protein Msed_0328

Msed0341 1.248345 3.497665 -2.24932 7.391742 hypothetical protein Msed_0341

Msed0347 1.482334 2.788498 -1.30616 9.78248 hypothetical protein Msed_0347

Msed0380 0.719314 2.043622 -1.32431 5.655999 hypothetical protein Msed_0380

Msed0429 -1.11448 0.319754 -1.43423 2.985354 hypothetical protein Msed_0429

Msed0450 1.529118 0.532163 0.996955 3.204346 protein of unknown function DUF1291 Msed0456 1.966517 0.985354 0.981163 3.333186 hypothetical protein Msed_0456

Msed0458 1.739429 2.913814 -1.17439 5.319824 hypothetical protein Msed_0458 bacterial adhesion domain

Msed0467 -1.62851 -0.16974 -1.45877 8.036316 major facilitator superfamily MFS_1

Msed0478 4.462937 2.754169 1.708768 7.290496 hypothetical protein Msed_0478 cytochrome b558-566

Msed0479 4.402287 1.956774 2.445513 8.088448 hypothetical protein Msed_0479 hypothetical membrane/ extracellular protein

Msed0480 4.772978 2.858172 1.914805 10.47886 cytochrome c oxidase, subunit II quinol oxidase (subunit 2)

Msed0482 2.370005 1.299184 1.070821 3.460972 hypothetical protein Msed_0482 hypothetical protein

Msed0500 0.890202 2.481572 -1.59137 4.070089 Cytochrome b/b6, N-terminal domain quinol oxidase (cyt b subunit) Msed0504 0.802073 1.820359 -1.01829 1.671054 hypothetical protein Msed_0504 cytochrome b558-566 (subunit A)

(Table 2.A1 continued)

Msed0507 -0.50559 1.329035 -1.83463 5.611987 pyruvate/ketoisovalerate oxidoreductase, gamma subunit Msed0508 -0.1428 1.727612 -1.87042 5.83803 pyruvate ferredoxin/flavodoxin oxidoreductase, delta subunit Msed0509 -1.04761 0.952397 -2 5.462319 pyruvate flavodoxin/ferredoxin oxidoreductase domain protein Msed0510 0.275727 2.281246 -2.00552 6.278337 thiamine pyrophosphate enzyme domain protein TPP-binding Msed0511 -1.22357 0.439401 -1.66297 6.035386 Radical SAM domain protein Msed0512 -1.51815 0.111755 -1.6299 4.338534 Radical SAM domain protein Msed0513 -1.8879 0.145947 -2.03385 6.475101 beta-lactamase domain protein Msed0514 -1.62661 0.29237 -1.91898 5.977738 peptidase U32 Msed0515 -2.24367 0.236966 -2.48063 9.396525 Radical SAM domain protein Msed0516 2.259373 3.22228 -0.96291 7.308375 hypothetical protein Msed_0516 conserved hypothetical protein Msed0518 -0.5825 2.868242 -3.45074 11.29534 hypothetical protein Msed_0518 Msed0529 -0.34994 0.811967 -1.16191 4.510092 hypothetical protein Msed_0529 Msed0535 -2.058 0.316775 -2.37477 12.4618 hypothetical protein Msed_0535 Msed0562 0.723143 -0.51668 1.239818 3.0977 hypothetical protein Msed_0562 Msed0578 1.620772 0.497995 1.122777 3.26936 hypothetical protein Msed_0578 Msed0579 4.622114 3.098818 1.523296 5.042805 hypothetical protein Msed_0579 Msed0621 1.367171 0.137071 1.2301 4.198937 CopG domain protein DNA-binding domain protein Msed0705 0.589696 1.953677 -1.36398 4.682442 peptidase S8 and S53, subtilisin, kexin, sedolisin Msed0960 -2.95615 -1.61875 -1.33739 9.290592 Uroporphyrin-III C/tetrapyrrole (Corrin/Porphyrin) methyltransferase uroporphyrin-III C-methyltransferase Msed0961 -2.43912 -0.87559 -1.56353 8.452756 nitrite and sulphite reductase 4Fe-4S region sulfite reductase putative adenosine phosphosulfate (APS) Msed0962 -0.64701 1.033341 -1.68035 5.859812 adenylylsulfate reductase, thioredoxin dependent reductase Msed1001 -2.07996 -1.03373 -1.04623 9.128675 major facilitator superfamily MFS_1 Msed1003 -0.23692 0.805846 -1.04276 3.78879 Methyltransferase type 11 Msed1020 -2.35739 -0.67069 -1.6867 7.366439 hypothetical protein Msed_1020 Msed1095 -1.7796 -0.65482 -1.12478 4.452538 major facilitator superfamily MFS_1

(Table 2.A1 continued)

Msed1181 -1.92615 -0.52462 -1.40153 7.923546 extracellular solute-binding protein, family 1 Msed1189 1.452693 4.07888 -2.62619 7.486677 hypothetical protein Msed_1189 Msed1191 -1.00596 1.087282 -2.09325 4.013792 Rieske (2Fe-2S) domain protein fused cyt b556 and Rieske protein archaeal flagellin / bacterial Type II/IV N-terminal Msed1193 -0.13844 4.433908 -4.57235 14.83607 hypothetical protein Msed_1193 domain protein Msed1194 -0.34969 3.650802 -4.00049 13.61472 hypothetical protein Msed_1194 hypothetical membrane/extracellular protein Msed1195 -1.85531 2.278565 -4.13388 10.82616 hypothetical protein Msed_1195 hypothetical membrane/extracellular protein Msed1196 -0.93853 3.493232 -4.43176 13.74616 hypothetical protein Msed_1196 hypothetical membrane/extracellular protein Type II/IV secretion system protein (ATP-binding Msed1197 -1.45319 2.178875 -3.63207 11.36891 type II secretion system protein E subunit) Msed1198 -0.48574 2.094294 -2.58003 6.271236 type II secretion system protein Type II/IV/flagellar subunit (FlaJ) Msed1205 0.324324 -0.70184 1.026161 3.575471 hypothetical protein Msed_1205 Msed1273 2.639167 1.629851 1.009316 2.976707 hypothetical protein Msed_1273 Msed1355 -0.74958 1.345537 -2.09512 9.902953 major facilitator superfamily MFS_1 Msed1370 2.829117 3.802375 -0.97326 3.421444 hypothetical protein Msed_1370 Msed1403 0.640745 -0.38195 1.022692 5.015901 putative endoribonuclease L-PSP Msed1407 0.276398 -0.81722 1.093618 2.242475 hypothetical protein Msed_1407 Msed1409 0.826514 -0.16827 0.994781 2.078845 hypothetical protein Msed_1409 Msed1455 1.952647 0.985069 0.967579 2.102073 amino acid permease-associated region amino acid transporter / permease Msed1461 0.657536 1.760646 -1.10311 4.20919 hypothetical protein Msed_1461 conserved hypothetical membrane protein Msed1520 -1.26003 -0.2939 -0.96613 2.379938 hypothetical protein Msed_1520 conserved hypothetical protein hypothetical ABC transporter (membrane Msed1528 0.875498 1.828946 -0.95345 4.174566 protein of unknown function DUF214 component) Msed1529 -0.31693 0.85714 -1.17407 7.006232 hypothetical protein Msed_1529 conserved membrane protein Msed1578 1.868492 0.840441 1.028051 3.970448 purine or other phosphorylase, family 1 S-methyl-5-thioadenosine phosphorylase Msed1580 2.80297 1.131568 1.671401 10.46485 hypothetical protein Msed_1580 conserved hypothetical protein Msed1607 1.078738 0.005388 1.07335 3.650783 Pyridoxal-5'-phosphate-dependent enzyme, beta subunit hypothetical cysteine synthase A

(Table 2.A1 continued)

Msed1612 0.315955 -0.87125 1.187204 4.363882 glutamine synthetase, type I glutamine synthetase Msed1636 3.694055 2.695301 0.998754 2.79924 ribosomal protein L10 50S / 60S acidic ribosomal protein L10 Msed1668 0.941151 2.168978 -1.22783 5.507937 Phosphopyruvate hydratase enolase Msed1732 3.916164 2.84632 1.069844 3.162545 hypothetical protein Msed_1732 conserved hypothetical protein Msed1865 2.880897 1.909626 0.971271 1.589196 ribosomal protein L10E 50S ribosomal protein L10E Msed1892 0.639577 -0.34445 0.984025 2.195222 hypothetical protein Msed_1892 conserved hypothetical protein Msed1910 2.467373 1.422847 1.044527 2.116246 hypothetical protein Msed_1910 conserved hypothetical protein Msed1985 3.300543 2.099095 1.201448 2.536002 hypothetical protein Msed_1985 conserved hypothetical protein Msed2028 1.838421 0.766336 1.072085 2.814634 transcriptional regulator, AbrB family hypothetical vapB antitoxin Msed2132 4.403605 3.239615 1.16399 3.845482 ribosomal protein L7Ae/L30e/S12e/Gadd45 50S ribosomal protein L7Ae Msed2164 1.505103 5.022284 -3.51718 17.01174 protein of unknown function DUF87 hypothetical ATP-binding protein Msed2211 2.430599 1.368499 1.0621 3.139797 RNA polymerase Rbp10 Msed2252 2.818539 1.826555 0.991984 2.719227 ribosomal protein L37e nc_mse0319 0.071723 -1.00796 1.079685 3.628412 nc_mse1148 -0.32922 1.050516 -1.37974 4.431681 nc_mse1780 -0.13491 0.930316 -1.06522 8.820882

Chapter 3

Components of electron transport chains in the extremely thermoacidophilic crenarchaeon Metallosphaera sedula identified through iron and sulfur compound oxidation transcriptomes

Kathryne S. Auernik and Robert M. Kelly

Department of Chemical and Biomolecular Engineering North Carolina State University Raleigh, NC 27695-7905

Published in Applied and Environmental Microbiology 74(24): 7723-7732 (2008)

70 ABSTRACT

The crenarchaeal order Sulfolobales collectively contains at least five major terminal oxidase complexes. Based on genome sequence information, all five complexes are found only in Metallosphaera sedula and Sulfolobus tokodaii, the two sequenced Sulfolobales capable of iron oxidization. While specific respiratory complexes in certain Sulfolobales have been characterized previously as proton pumps for maintaining intracellular pH and generating proton motive force (pmf), their contribution to sulfur and iron biooxidation has not been considered. For M. sedula growing in the presence of ferrous iron and reduced inorganic sulfur compounds (RISCs), global transcriptional analysis was used to track the response of specific genes associated with these complexes, as well as other known and putative respiratory electron transport chain elements. ORFs from all five terminal oxidase or bc1-like complexes were stimulated on one or more conditions tested. Components of the fox (Msed0467-0489) and soxNL-cbsABA (Msed0500-0505) terminal/quinol oxidase clusters were triggered by ferrous iron, while the soxABCDD’ terminal oxidase cluster

(Msed0285-0291) were induced by tetrathionate and S°. Chemolithotrophic electron transport elements, including a putative tetrathionate hydrolase (Msed0804), a novel polysulfide/sulfur/DMSO reductase-like complex (Msed0812-0818), and a novel heterodisulfide reductase-like complex (Msed1542-1550), were also stimulated by RISCs.

Furthermore, several hypothetical proteins were found to have strong responses to ferrous iron or RISCs, suggesting additional candidates in iron or sulfur oxidation-related pathways.

From this analysis, a comprehensive model for electron transport in M. sedula could be

71 proposed as the basis for examining specific details of iron and sulfur oxidation in this bioleaching archaeon.

INTRODUCTION

Certain extremely thermoacidophilic archaea are promising candidates for biomining operations targeting the recovery of base and precious metals (44).These microorganisms grow at elevated temperatures where abiotic ferrous oxidation rates are accelerated and where passivation of mineral surfaces from reduced inorganic sulfur compounds (RISCs) is minimal (44).To tap into the biotechnological potential of these microorganisms their physiological characteristics that relate to metal mobilization need to be better understood, especially mechanisms underlying biooxidation of iron (Fe2+) and reduced inorganic sulfur compounds (RISCs). Biooxidation implicates membrane-associated protein complexes that mediate electron transport, which seemingly determine the capability and capacity of extreme thermoacidophiles for metal and sulfur mobilization. However, the complexes required for bioleaching have not been established. Models that address the specific and collective function of electron transport complexes in the Sulfolobales are needed to provide a physiological framework for exploring the intricacies of biological metal and sulfur oxidation.

Current knowledge of respiratory electron transport chains (ETC) in the Sulfolobales is based primarily on separate biochemical characterization studies performed in various members of this order (Figure 3.1, Table 3.1). Based on studies in Acidianus ambivalens,

Sulfolobus metallicus, and Sulfolobus tokodaii (3, 17, 24, 32, 40), electrons from NADH or

72 succinate oxidation presumably enter ETCs via NADH:quinone oxidoreductases (NDH-II) or succinate:quinone oxidoreductases (SdhABCD, Type E). Reduced quinones (typically caldariella or sulfolobus-type quinones) then deliver electrons to one of several terminal oxidase complexes, composed of cytochrome oxidase and/or bc1 complex-like subunits.

These subunits are related to aa3 terminal oxidase complexes in A. ambivalens

(DoxBCE)(14, 37, 42) and Sulfolobus acidocaldarius (SoxABCD+SoxL)(16, 34, 35), the bb3 terminal oxidase complex in S. acidocaldarius (SoxEFGHIM)(11, 29, 33), the putative

FoxAB terminal oxidase in S. metallicus (the recent discovery of which coincided with the first report of ferrous iron oxidation in S. tokodaii) (5), and bc1-complex in S. acidocaldarius

(SoxLN)(22). Both FoxAB and SoxNL are associated with cytochrome b558/566–like proteins

(FoxCD and CbsAB), which exhibit characteristics found in other terminal oxidase subunits

(i.e., glycosylated like SoxBC, conformational flexibility like Rieske proteins) and are probably involved in ETC in a similar manner (5, 21, 22, 37, 47). Terminal oxidase complexes are important in extreme thermoacidophiles as proton pumps for maintaining intracellular pH and generating proton motive force (pmf). Other proteins may contribute to pmf generation by using electrons from Fe2+ and RISCs to reduce quinones. Leading candidates for this function include the thiosulfate:quinone oxidoreductase (TQO) in A. ambivalens (37), as well as the tetrathionate hydrolase (TetH) in the mesoacidophilic bioleaching bacteria Acidithiobacillus caldus and Acidithiobacillus ferrooxidans (9, 26, 46).

Recent work with A. ferrooxidans has shown that the respiratory ETC encoded in the rus and petI operons are involved in uphill electron transport from Fe2+ oxidation, with electrons being donated via the blue copper protein, rusticyanin, to a bc1 complex operating in reverse, possibly to a NADH:quinone oxidoreductase (8, 15, 23). However, the recently

73 completed genome sequence of the bioleacher Metallosphaera sedula revealed that these operons are not present (2, 12). Although some individual components are similar (i.e., blue copper protein and PetAB-like subunit sequences), no capacity for uphill electron transfer has been demonstrated to date in the Sulfolobales.

Functional genomics approaches are potentially useful for gleaning the detailed features of respiratory ETC chains with respect to biological iron and sulfur oxidation, since genes encoding ETC components in both mesoacidophilic and extremely thermoacidophilic bioleachers apparently are differentially transcribed in the presence of RISC- or Fe2+-based substrates (5, 8, 27, 43, 51). Utilizing this observation, the global transcriptional response of bioleacher M. sedula to the presence and/or absence of inorganic electron donors was investigated during growth on yeast extract. Transcriptome response was used to support the role of previously characterized proteins in ferrous iron and RISC-induced ETCs and to propose new candidates for members of ETCs. The model resulting from this analysis provides the basis for probing metabolic features of iron and sulfur oxidation in extreme thermoacidophiles.

MATERIALS AND METHODS

Growth of M. sedula in the presence of inorganic iron and sulfur compounds.

M. sedula (DSMZ 5348) was grown aerobically at 70oC in an orbital shaking oil bath at 70 rpm on DSMZ 88 medium (pH 2), supplemented with 0.1% yeast extract (YE, “Y”). Growth medium was supplemented with 10g/L of FeSO4·7H2O (1% w/v, 36mM, “YFS”), 20g/L of

o elemental sulfur (S , “YS”) (2% w/v), 10g/L K2S4O6 (1% w/v, 33mM, “YKT”), or 6g/L K2SO4

(29mM, “YKS”). Cells were acclimated to supplemented media through 3 passages before harvesting. On the 4th passage, an initial density of mid 106 cells/mL was used in two 1 L bottles containing 300 mL volume (four 1L bottles were used for FeSO4-supplemented condition). Cultures in mid-exponential phase (~24 h post-inoculation, mid-107 cells/mL) were harvested by quickly chilling and then centrifuging at 9510 x g for 15 minutes at 4oC.

M. sedula oligonucleotide microarray construction and transcriptional response analysis. A spotted whole genome oligonucleotide microarray based on at least

2256 protein-coding ORFs was constructed (2328 probes x 5 replicates = 11640 spots per array), as described previously (2). Probes (60-mers) were designed using OligoArray 2.1 software (45) and synthesized by Integrated DNA Technologies (IDT, Coralville, IA).

RNA was extracted and purified (RNAqueous, Ambion), reverse transcribed

(Superscript III, Invitrogen), re-purified, labeled with either Cy3 or Cy5 dye (GE Healthcare), and hybridized to one of five microarray slides (Corning). Slides were scanned on a Perkin-

Elmer scanner and raw intensities were quantitated using ScanArray® Express 2.1.8 software. Normalization of data and statistical analysis were performed using JMP

Genomics 3.1 software (SAS, Cary, NC). Raw data, as well as final log2 fold changes are listed in NCBI’s Geo under series accession number GSE12044. In general, significant differential transcription, or “response”, was defined to be fold changes at or above 2

(where {log2,=|±1|} = 2-fold change) with significance values at or above the Bonferroni correction, which was 5.4 (equivalent to a p-value of 4.0*10- 6) for this data set.

Comparative genomics analysis. For comparative genomics, M. sedula gene sequences and annotations were downloaded from the JGI microbial genome website

(http://genome.ornl.gov/microbial/msed/28feb07/). Other sequences referenced were obtained from GenBank, with the exception of A. ferrooxidans ATCC 23270 sequences, which were obtained from The Institute for Genome Research’s Comprehensive Microbial

Resource (TIGR CMR). The Basic Local Alignment Search Tool (BLAST) searches were conducted using a BLOSUM62 matrix with either the NCBI protein-protein blast program

(BLASTP) against the Swissprot database, nr database, or Metallosphaera, three

Sulfolobus, and Ferroplasma genomes, the ORNL Microbial BLAST server’s BLASTP program against the msed database, or the CMR tblastn program against the A. ferrooxidans genome.

RESULTS

Genome sequence analysis was used in conjunction with transcriptomics to examine the presence and potential function of ETC in M. sedula. Previous reports on the biochemical function of specific proteins implicated in electron transfer in other extremely thermoacidophilic archaea (and mesoacidophiles as well) were incorporated into the analysis. From this, a model could be proposed for ETC that included new annotations for putative proteins in the in M. sedula genome sequence.

Quinone reduction from organic electron donors. Other than the efforts with

NADH:quinone oxidoreductases (NDH-II) mentioned above, little work has been done with

Type I NADH dehydrogenases (NDH-I) in extremely thermoacidophilic archaea. In fact, the lack of ORFs with similarity to NADH and FMN binding regions in NDH-I enzymes suggests that the actual electron donor may not be NADH (40). Type III NDHs have not been found in extremely thermoacidophilic archaea, and Type IV NDHs (NDH-IV) have just recently been discovered in archaea (39). The best candidates for NDH-I subunits

(NuoACDHIJKLN, Msed1895-1903), NDH-II (Msed2059), and NDH-IV (Msed0428) in M. sedula were not differentially transcribed during growth in the presence of Fe2+ or RISCs. In addition to succinate dehydrogenase (SdhABCD,) encoded by Msed0674-7, another appears to be encoded by Msed1112-6 (SdhACD-FumAB), which has two iron-sulfur subunits more similar to class I fumarase than SdhB.

Neither locus responded to Fe2+ or RISCs. Because ORFs encoding quinones in members of the Sulfolobales have yet to be annotated, transcriptional response information is not readily available.

Quinone reduction via inorganic electron donors. The M. sedula genome encodes a putative TetH (Msed0804) (2); in fact, Msed0804 was up-regulated on both

RISC-supplemented conditions, YKT (at 32-fold this was the largest change noted in this data set) and YS (see Table 3.2 and Table 3.A1). Genes encoding mesoacidophile TetHs

2- o (AfeTetH (10, 26) and AcaTetH (9, 46)) were also up-regulated on S4O6 .and S , although

AcaTetH induction was less conclusive since So concentrations were 4-fold lower than those used for A. ferrooxidans and M. sedula studies (5g/L vs. 20 g/L). AcaTetH contains a pyrrolo-quinoline quinone (PQQ) binding motif, shown to interact with a quinone (46).

Msed0804 also contains a PQQ-binding motif, presenting the possibility that it donates

2- electrons to quinol oxidases (bc1 complexes). TQO (DoxDA), which oxidizes S2O3 to

2- S4O6 , connects to ETC in a similar manner (37). M. sedula encodes a putative TQO

(Msed0363-4) with a separate DoxD-like sequence (Msed0374). Because of TQO’s RISC oxidation function, it was hypothesized that transcription of these subunits would be higher in the presence of RISCs. However, these ORFs in M. sedula did not respond to any conditions tested (Table 3.A1).

New components of Fe(II)-induced electron transport chains. A blue copper protein, rusticyanin, is implicated in electron transport from iron oxidation and one version of which has been extensively characterized in A. ferrooxidans(4, 51, 53). The M. sedula genome encodes four blue copper protein-like sequences: two sulfocyanins (Msed0323 and

Msed0826) and two rusticyanins (2). However, none of these ORFs responded to any significant extent for any of the conditions tested (Table 3.A2). In most cases, levels for these ORFs were slightly below average (lsm < 0) when compared to all transcripts, with

Msed0323 > Msed1206 > Msed0966 > Msed0826 for all conditions tested (Figure 3.A1).

New components of RISC-induced electron transport chains. The Msed0810-

0818 locus was stimulated on YS and YKT (see Figure 3.2, Table 3.2, and Table 3.A3).

With the exceptions of Msed0811, Msed0813, and Msed0818, all ORFs in this locus were up-regulated at least 2-fold on the RISCs, with maximum induction of Msed0812 on YKT vs.

YFS (18-fold). Homologous putative proteins, found similarly clustered in S. solfataricus, S. tokodaii, and S. acidocaldarius (Table 3.A4), belong to the dimethyl sulfoxide (DMSO) reductase family, which includes thiosulfate reductase (PhsABC) from Salmonella typhimurium (1, 20), polysulfide reductase (PsrABC) from Wolinella succinogenes (30),

78 chlorate reductase (ClrABC) from Ideonella dechloratans (50), dimethyl sulfide dehydrogenase (DdhABC) from Rhodovulum sulfidophilum (36), and sulfur reductase

(SreABC) from A. ambivalens (31). This family of proteins is mostly found in anaerobic bacteria, although anoxic conditions appear to be required for transcriptional regulation, but not activity (1, 20). Members of this family are heterotrimers in which the catalytic α and β subunits are both hydrophilic and contain 4Fe-4S and/or 3Fe-4S coordination sites (as recognized by characteristic Cys-rich motifs). The α subunit also binds a Mo co-factor, specifically molybdopterin guanine dinucleotide (bisMo). A chaperone protein, often encoded between the β and γ subunits, helps in proper folding/assembly of the α and β subunits. The hydrophobic γ subunit functions as the membrane anchor. Msed0814-0815 encodes the equivalents of the α (only bacterial N terminal domain (NTD) conserved) and β subunits, respectively, while Msed 0816 is related to a TorD/DmsD-like chaperone/assembly protein, specifically Aam SreE, Ide ClrD, and Rsu DdhD. Msed0812, a predicted transmembrane protein on the opposite strand from the rest of the Msed0810-

0818 cluster, is related to the membrane anchors Wsu PsrC and A. ferrooxidans SreC

(Afe_0918) (membrane anchor sequences have been reported to be less conserved than the catalytic subunits (50)) and also appears to be similar to formate-dependent nitrate reductase membrane anchor proteins (NrfD-like, COG3301). In some cases the γ subunit is thought to be a cytochrome b involved with electron transfer (36). However, Msed0812 has no similarity to any cytochrome b encoded elsewhere in its genome. Msed0817 is homologous to Aam SreD, thought to be an FeS-binding electron transfer protein involved with SreABC. The remaining 3 members of the cluster are hypothetical proteins: one with weak homology to a transcriptional regulator domain possibly involved with the regulation of

79 this gene cluster’s transcription (Msed0810, COG1414), the second with weak homology to a GTP-binding domain and ATP-dependent helicase/nuclease which may be associated with the bisMo cofactor synthesis (Msed0811), and the third a tetra-tri-co-peptide repeat

(TPR) domain which has been associated with assembly of multi-subunit proteins

(Msed0813, cd00189).

The Msed1540s region was found to be differentially transcribed on YS and YKT compared to YFS (Figure 3.2, Table 3.2, and Table 3.A5). With the exception of Msed1547, all ORFs in this locus were up-regulated at least 2.0-fold, with an overall stronger transcriptional response for YKT than for YS. Maximum induction for this cluster was

Msed1549 on YKT vs. YFS (19-fold). Although not differentially transcribed, Msed1547 was highly transcribed on all conditions tested, with an lsm ranging from 3.4 to 4.4 compared to the experimental loop’s normalized average gene expression level of 0.0. As opposed to most of the Msed0810-0818 region, the Msed1542 region appears to be transcribed at higher than average levels on Y alone. RISCs boosted transcription further, while Fe2+ led to a slight decrease for the four ORFs compared to Y alone (data not shown).

ORFs related to the 1540s region are all found similarly clustered in S. solfataricus,

S. tokodaii, and S. acidocaldarius (see Table 3.A6). The mesoacidophilic bioleaching bacterium, A. ferrooxidans, and hyperthermophilic bacterium, Aquifex aeolicus, also contain a similar cluster. The most-studied similar sequences, however, are the heterodisulfide reductases (HdrABC) found in the methanogenic archaea (18, 19).

ORFs 1542-4 overlap with one another (by 4 and 14bp, respectively). Although

Msed 1544 is not similar to previously characterized proteins, Msed 1542 (306aa) and the

80 central region of Msed1543 (100aa out of 230aa) are related to the HdrB and HdrC subunits of several mesophilic and hyperthermophilic methanogens. Msed1546-7 appear to encode additional HdrBC subunits (444 and 280aa, respectively), but have slightly less homology to the same methanogen HdrBC subunits than Msed1542-3. Interestingly, both

M. sedula HdrC-like sequences (Msed1547 & 1543) share only 27% identity in their N- terminal domains (NTD), which contains Cys-rich motifs typically corresponding to Fe-S binding regions, while the two HdrB-like sequences (Msed 1546 and 1542) are not similar.

Msed1545 (365aa) is similar to the NTD and central regions of HdrA sequences of the same methanogens. None of these methanogens contain hdrA in the same neighborhood as hdrBC, although Methanothermobacter marburgensis reportedly contains an hdrACB gene organization comparable to that found in M. sedula(18). Msed1548 (80aa) is related to a SirA family protein, part of a two-component response regulation system in bacteria

(IPR001455), and COG0425, predicted redox protein regulator in disulfide bond formation.

This implicates Msed1548 in regulation of the Msed1540s Hdr-like region. Msed1549 and

1550 both lie upstream of this possible regulator, with Msed1550 on the opposite strand from the rest of the 1540s region. Msed1549 has no homology to any characterized protein.

While Msed1550 is annotated as a DsrE-family protein involved with intracellular sulfur reduction (IPR003787), it is not similar to DsrE of the dsr locus in Allochromatium vinosum(41).

Quinone oxidation via terminal oxidase complexes. The SoxABCL terminal oxidase cluster is based on both quinol oxidase (SoxCL) and cytochrome oxidase (SoxAB) components (2, 16, 35). As expected (2, 27), soxABCL, Msed0288-0291, was significantly

81 up-regulated on both YS and YKT (15-fold for Msed0285) compared to YFS (Figure 3.2,

Table 3.2, Table 3.S7). This is consistent with previous reports of differential expression of terminal oxidase subunits as a function of substrate (27). Several ORFs upstream of soxABCL (Msed0283-0286, each encoding putative proteins of ~100-150aa) showed transcription patterns similar to soxABCL. Homologs to Msed0283-0284 are present in S. solfataricus (Sso2640-1) and S. tokodaii (ST1697-8), although they are not co-located with the SoxABCL cluster. Msed0285-6 are predicted membrane proteins whose homologs are co-located with the soxABCL clusters present in S. solfataricus (Sso2656-2662), S. tokodaii

(ST0132-0137), and S. acidocaldarius (Saci2087-2092). This suggests that they are associated with the SoxABCL terminal oxidase complex, and possibly the two small polypeptides (one named SoxD) that purified with SoxABC in S. acidocaldarius (35). These polypeptides co-migrated on a gel at 14kDa (35); both Msed0285 and Msed0286 have predicted product masses of ~14.5 kDa. SoxD was reported to be transcribed at the end of the soxABC operon, while the other had a partial translation product, which did not match known S. acidocaldarius sequences (35). Using the S. acidocaldarius genome, the unknown translation product can now be aligned with a segment of the NTD of Saci_2091, whose top hit in the M. sedula genome is Msed0286 (SoxD’). Msed0285 is similar to

Saci_2092, likely encoding the originally described SoxD.

The DoxBCE terminal oxidase complex was originally isolated from A. ambivalens grown in the presence of So (42). Table 3.A8 shows that M. sedula doxBCE (Msed2030-2) was not significantly induced by RISC substrates, although normalized transcription levels

(lsms) indicate that these subunits were most constitutively transcribed under all conditions tested. Note that a separate doxB-like gene (Msed0570) was up-regulated on both RISC

82 substrates. A separate doxB locus was also identified in A. ambivalens, although its function has not been confirmed (42).

The SoxEFGHIM terminal oxidase complex was originally isolated and studied in S. acidocaldarius (11, 29, 33). More recent studies in M. sedula showed soxM to be up-

o regulated on Y compared to Y+S or Y+ pyrite (FeS2). However, it was not clear whether this differential transcription was triggered by the presence of Fe(II) and RISCs or the decrease in Y from 0.1% to 0.02% (27). Here, when the concentration of Y was held constant for all conditions tested, significant up-regulation of soxM was seen only on Y vs

YFS (Table 3.A8). Interestingly, the cytochrome b subunit transcript, soxG (Msed0321), was up-regulated on YFS (2- to 3-fold) compared to Y, YKT, and YS (Table 3.A8).

Examination of the gene neighborhoods of sequenced Sulfolobales shows that, unlike soxABC and doxBCE, soxHGM are not co-located on the same strand; soxM is located on the strand opposite soxHG in S. acidocaldarius, S. solfataricus, S. tokodaii, and M. sedula.

The SoxNL-CbsAB and FoxABCD clusters were originally described in S. acidocaldarius (22) and S. metallicus (5), respectively. Work in M. sedula showed that cbsA is up-regulated on pyrite compared to Y or So (27), while work in S. metallicus showed that foxABCD is up-regulated on Fe(II) vs. So(5). Preliminary Y+Fe(II) vs. Y transcriptional response analysis of M. sedula’s soxLN-cbsAB and foxABCD clusters showed soxLN- cbsAB and the first copy of foxA (Msed0484) was up-regulated on Y+Fe(II) vs Y, but foxBCD was down-regulated under the same conditions. The second copy of foxA (foxA’,

Msed0485) showed no significant differential transcription (2). Here, when M. sedula was exposed to higher amounts of Fe(II) for longer periods of time, soxN-cbsAB and the first copy of foxA were significantly up-regulated on YFS vs. Y, while foxB and foxA’ were

83 significantly down-regulated (2-fold and 15-fold, respectively) under the same conditions

(Table 3.A8). Transcripts from soxL and foxCD (CbsAB-like sequences) showed no significant differential transcription under these conditions. Normalized transcription levels for foxAA’BCD were all above average, with 70% of foxAA’BC lsms ranging from 2.0-5.2, indicating that these genes are highly transcribed under all conditions tested. Interestingly, foxA maximum transcription levels occurred on YFS, the same conditions under which foxA’ minimum transcription levels occurred (see Figure 3.A2). Also of note, Msed0502 is listed as a pseudogene in GenBank; however, microarray probes for this ORF (nc_mse1147 & nc_mse1148) showed significant up-regulation on YFS vs. Y and RISCs (Figure 3.2 and

Table 3.A8) and on YFS vs. Y (from (2); see GEO accession# GSE11296). The translated

Msed0502 draft sequence fragments share similarities with CbsA cytochromes b, like the

CbsA encoded by Msed0504. Although microarray probe design aims to avoid cross- hybridization, it was verified via an independent nucleotide BLAST search of the M. sedula genome that probes for Msed0502 hybridize fully with sections of Msed0502 only, and not

Msed0504.

Fe(II) and RISC-induced hypothetical proteins. In some cases, ORFs encoding hypothetical or conserved hypothetical proteins responded to the tested conditions, by as much as 13-fold for Msed1905 on YS vs. YFS (Table 3.A9). In some instances the differentially transcribed ORFs may be involved with the respiratory electron transport chains (i.e., Msed0509, a subunit of a pyruvate flavodoxin/ferredoxin oxidoreductases complex), but in most cases there is insufficient annotation to suggest a respiration-related function. These ORFs merit further investigation.

DISCUSSION

With the sequencing of the first genome from an extremely thermoacidophilic bioleacher, it has become evident that M. sedula does not contain the same iron oxidation pathways as those previously studied in the mesoacidophile A. ferrooxidans. Little or no previous work has been done on the iron oxidation pathways in extremely thermophilic bioleachers, but potential candidates in these pathways can be proposed from transcriptome and bioinformatic analysis, including novel components with no similarity to other proteins with known iron oxidation functions. A similar situation exists with some aspects of sulfur oxidation, although an arguably larger body of literature exists on bacterial sulfur metabolism.

The complexity of sulfur chemistry presents a significant challenge to understanding

2- archaeal inorganic sulfur metabolism (28). For example, both S° and tetrathionate (S4O6 )

2- contain S-S bonds with S in the zero oxidation state. However, S4O6 also contains S in the

+5 oxidation state, which potentially impacts the S-responsive genes in the M. sedula transcriptome. Both RISCs are subject to abiotic disproportionation reactions, although these are unfavorable at pH < 4 (28). However, there is a possibility that small amounts of

2- 2- o thiosulfate (S2O3 ), and sulfide (S ) in the case of S , may also be formed. Furthermore,

2- 2- o S2O3 at pH <4 forms sulfite (SO3 ) and S . Thus, despite the relatively high purity of RISC

o 2- 2- 2- supplements, it is possible that M. sedula experienced sulfur as S , SO3 , S , and S2O3 .

o 2- 2- In addition to the abiotic production of S , SO3 , and S2O3 , TetH may catalyze a

2- o - disproportionation reaction, splitting S4O6 into other sulfur compounds, namely S , HSO3

2- and SO4 (28). Experimental data suggest that the membrane-bound TetH in A.

2- - ferrooxidans produces S2O3 instead of HSO3 (26), and that the periplasmic TetH in

2- 2- - Acidithiobacillus caldus produces S2O3 and S5O6 instead of HSO3 (46). Reactions like

2- 2- these could potentially add significant amounts of S2O3 and S5O6 RISCs to the M. sedula media, a possibility that needs to be considered when interpreting transcriptome information.

By utilizing the same amount of complex organic substrate in all conditions tested

(Y=0.1%), the experimental loop used here was designed to eliminate differential transcription caused by the type or concentration of heterotrophic substrate present. This was done so that the focus would be on response to ferrous iron or RISCs (inorganic electron donors). No significant differences in growth rate/yield were observed for any conditions tested. However it must also be considered that the presence of a heterotrophic substrate may impact the response to Fe(II) and/or RISC differently than under autotrophic conditions. For example, the response of the fox gene cluster in S. metallicus (an autotroph) (5) may be different than in M. sedula growing under heterotrophic conditions

(this work and (2)). While more details of M. sedula autotrophy have recently become available (6), the influence of autotrophy, heterotrophy, or even mixotrophy on bioleaching associated functions such as iron and sulfur oxidation is not known.

The role of blue copper protein, rusticyanin, as an intermediate Fe(II)-sourced electron transfer protein between cytochromes c (Cyc2 and Cyc1 or CycA1) has been studied in A. ferrooxidans (8, 51-53), but the significance of rus-like sequences in extremely thermoacidophilic archaeal bioleachers is unclear. The presence of blue copper protein, sulfocyanin, in M. sedula is expected, as it has already been studied in S. acidocaldarius

(11, 29). Since the proposed role of both of these blue copper proteins involves electron transfer, it is somewhat surprising to see no differential response of these four ORFs in M. sedula transcriptional studies.

The putative DMSO reductase-like enzyme complex (Msed0810-0818) would not be expected to involve sulfur as a terminal electron acceptor, since all M. sedula cultures involved were grown aerobically. A phylogram based on Aam SreA showed how different

DMSO reductase-like molybdoenzymes cluster (i.e. DMSO reductases distinct from sulfur reductases, distinct from formate dehydrogenases, etc.) (31). An expanded phylogram

(Figure 3.A3) shows that sequences encoding similar functions group together, with the exception of Aae1234 ( “DmsA” ), although this Aquifex aeolicus ORF is not based on experimental data showing DSMO as the most likely substrate. Msed0814 and its

Sulfolobus Mo-oxidoreductase homologs appear to group into a new subclade separate from the polysulfide and sulfur reductases, sharing a deep, but common root with the sulfide-oxidizing Rsu DdhA. Taken together with the transcriptional response from growth

o 2- on S and S4O6 , a catalytic function other than reduction of the sulfur substrate seems likely (i.e., RISC oxidation).

It is interesting that Msed1542-1550 encodes proteins with similarities to Hdr in methanogens. The role of Hdr in methanogens is to reduce the CoM-CoB heterodisulfide, regenerating both cofactors for further use in methanogenesis. M. sedula is neither known to contain cofactors CoM or CoB, nor participate in methanogenesis; therefore, HdrABC subunits in Msed1542-1550 region are believed to target a different substrate. Phylogenetic grouping appears to support this hypothesis (Figure 3.A4 shows a phylogram of HdrB-like sequences which are believed to contain the catalytic/active site for Hdr in M.

87 marburgensis(18)). The Msed1540s region is not predicted to contain transmembrane proteins, but the methanogen Hdr complex activity has been found in the membrane following gentle disruption (49). Alternative considerations should include association of the

Hdr complex with other transmembrane proteins outside the gene neighborhood or the possibility that some Hdr-associated subunits may be integral membrane proteins containing amphipathic helices not predicted with TMM software, as seen in the HdrB-like SucC sequence from A. ambivalens (32).

Comparative genomics has revealed that the sequenced Sulfolobales collectively contain almost all the terminal oxidase (or bc1 complex-quinol oxidase) complexes mentioned here. This raises the question as to why multiple terminal oxidases are needed

(while Sox M is a bb3 type, both SoxB and DoxB are aa3 types(40); FoxA is yet unknown).

The results presented here suggest that the relative presence of some terminal oxidase complexes is indirectly regulated by the presence of Fe(II) or RISCs. Also in M. sedula it has been noted that near gene duplications exist within some of these complex clusters

(i.e., cbsA: Msed0502 and 0504, foxAA’: Msed0484-5, and doxB: Msed0570 and 2032).

The purpose for near duplication is not clear, although in the case of cbsA and foxAA’, the difference in transcriptional profile suggests some differences in regulation, and thus the potential for functional distinction. It has been noted that while cytochromes b (a586), SoxC and SoxG, from S. acidocaldarius have significant sequence similarity, the redox potential of SoxG is considerably lower than that of SoxC, impacting electron donor/acceptor options

(16, 29). However, in Rubrivivax gelatinosus, similar Rieske subunits of the bc1 complex are known to substitute for each other to retain complex functionality (38).

One notable exception to the multiple terminal oxidase observation is the fox cluster

(containing soxHB-like and cbsAB-like sequences) present in S. metallicus, S. tokodaii, and

M. sedula (all shown to be iron oxidizers) and absent in S. solfataricus and S. acidocaldarius (no demonstrated iron oxidation capabilities). Considering this grouping, along with the observation that some fox sequences have low similarities to NADH or IMP dehydrogenase sequences (data not shown), suggests that the fox cluster may be the best candidate for uphill e- transport, should this pathway exist in extremely thermoacidophilic bioleachers. However, definitive work demonstrating the operation of this cluster in the reverse (uphill) direction would be required to support this hypothesis.

In addition to the A. ferrooxidans model for iron oxidation, other bacterial models are emerging. For mesoacidophilic Leptosprillum group II bacteria, Fe(II) electrons are hypothesized to be transferred to a cytochrome (cyt572) at the outer membrane and then by transported across the periplasm by a reddish cytochrome (cyt579) (48). In certain photosynthetic mesoneutrophiles, including Rhodobacter capsulatis, Rhodobacter strain

SW2, and Rhodopseudomonas palustris, iron oxidation is thought to occur via a putative periplasmic c-type cytochrome (FoxE or PioA) which transfers electrons to a pyrrolo- quinoline quinone domain protein or a high potential iron-sulfur protein,HiPIP (FoxY or

PioC) (13, 25). Both operons also contain a putative iron transport component (FoxZ and

PioB, respectively) that could facilitate access from the outer membrane to the periplasm, although biochemical work confirming specific function remains to be completed. While the current bacterial models share the common theme of a cytochrome c serving as the initial/primary electron acceptor, the thermoacidophilic archaeal model is likely to be different, as these organisms are not known to possess cytochromes c and also lack a true

89 periplasmic space. Interestingly, M. sedula has been reported to possess a yellow cytochrome capable of direct reduction by Fe(II) which exihibits a reduced absportion spectra peak at 572nm (7), the same wavelength reported for Leptospirillum group II bacteria’s proposed initial Fe(II) electron acceptor. Recent studies in M. sedula and S. metallicus have hypothesized that the 572nm absorption peak may be associated with either CbsA or the CbsA-like FoxC (5, 27), and previous characterization of CbsA in S. acidocaldarius suggested that it could serve an “ectoenzyme” function (21).

Summary. The availability of several Sulfolobales genome sequences has facilitated examination of respiratory ETC components. M. sedula’s capacity to oxidize iron and sulfur distinguish it from other sequenced Sulfolobales and currently limit the usefulness of comparative genomics in identifying previously unrecognized components of respiratory electron transport chains. However, for the first time, the transcriptional response of all the known components has been evaluated simultaneously and in some cases appears to be a function of potential chemolithotrophic substrate. This argues that these components (or their pre-transcriptional regulation) are important for oxidation of iron

(i.e., SoxNL-CbsAB, Fox cluster components) or RISCs (i.e., TetH, DMSO/sulfur reductase- like, Hdr reductase-like components). In addition, several hypothetical proteins with strong responses to Fe(II) and RISCs have been identified and will warrant further characterization to determine the nature of their involvement in Fe(II) and RISC oxidation. A key question going forward is how Fe(II) and RISC oxidation are impacted by autotrophic, heterotrophic, or mixotrophic modes of growth.

ACKNOWLEDGMENTS

KSA acknowledges an NIH T32 Biotechnology Traineeship for support. This work was also funded in part by a grant to RMK from the US National Science Foundation.

REFERENCES

1. Alami, N., and P. C. Hallenbeck. 1995. Cloning and characterization of a gene cluster, phsBCDEF, necessary for the production of hydrogen sulfide from thiosulfate by Salmonella typhimurium. Gene 156:53-7.

2. Auernik, K. S., Y. Maezato, P. H. Blum, and R. M. Kelly. 2008. The genome sequence of the metal-mobilizing, extremely thermoacidophilic archaeon Metallosphaera sedula provides insights into bioleaching-associated metabolism. Appl Environ Microbiol 74:682-92.

3. Bandeiras, T. M., C. A. Salgueiro, H. Huber, C. M. Gomes, and M. Teixeira. 2003. The respiratory chain of the thermophilic archaeon Sulfolobus metallicus: studies on the type-II NADH dehydrogenase. Biochim Biophys Acta 1557:13-9.

5. Bathe, S., and P. R. Norris. 2007. Ferrous iron- and sulfur-induced genes in Sulfolobus metallicus. Appl Environ Microbiol 73:2491-7.

6. Berg, I. A., D. Kockelkorn, W. Buckel, and G. Fuchs. 2007. A 3- hydroxypropionate/4-hydroxybutyrate autotrophic carbon dioxide assimilation pathway in Archaea. Science 318:1782-6.

7. Blake, R. C., 2nd, E. A. Shute, M. M. Greenwood, G. H. Spencer, and W. J. Ingledew. 1993. Enzymes of aerobic respiration on iron. FEMS Microbiol Rev 11:9- 18.

8. Bruscella, P., C. Appia-Ayme, G. Levican, J. Ratouchniak, E. Jedlicki, D. S. Holmes, and V. Bonnefoy. 2007. Differential expression of two bc1 complexes in the strict acidophilic chemolithoautotrophic bacterium Acidithiobacillus ferrooxidans suggests a model for their respective roles in iron or sulfur oxidation. Microbiology 153:102-10.

9. Bugaytsova, Z., and E. B. Lindstrom. 2004. Localization, purification and properties of a tetrathionate hydrolase from Acidithiobacillus caldus. Eur J Biochem 271:272-80.

10. Buonfiglio, V., M. Polidoro, F. Soyer, P. Valenti, and J. Shively. 1999. A novel gene encoding a sulfur-regulated outer membrane protein in Thiobacillus ferrooxidans. J Biotechnol 72:85-93.

11. Castresana, J., M. Lubben, and M. Saraste. 1995. New archaebacterial genes coding for redox proteins: implications for the evolution of aerobic metabolism. J Mol Biol 250:202-10.

12. Clark, T. R., F. Baldi, and G. J. Olson. 1993. Coal Depyritization by the Thermophilic Archaeon Metallosphaera sedula. Appl Environ Microbiol 59:2375- 2379.

13. Croal, L. R., Y. Jiao, and D. K. Newman. 2007. The fox operon from Rhodobacter strain SW2 promotes phototrophic Fe(II) oxidation in Rhodobacter capsulatus SB1003. J Bacteriol 189:1774-82.

14. Das, T. K., C. M. Gomes, T. M. Bandeiras, M. M. Pereira, M. Teixeira, and D. L. Rousseau. 2004. Active site structure of the aa3 quinol oxidase of Acidianus ambivalens. Biochim Biophys Acta 1655:306-20.

15. Elbehti, A., G. Brasseur, and D. Lemesle-Meunier. 2000. First evidence for existence of an uphill electron transfer through the bc(1) and NADH-Q oxidoreductase complexes of the acidophilic obligate chemolithotrophic ferrous ion- oxidizing bacterium Thiobacillus ferrooxidans. J Bacteriol 182:3602-6.

16. Gleissner, M., U. Kaiser, E. Antonopoulos, and G. Schafer. 1997. The archaeal SoxABCD complex is a proton pump in Sulfolobus acidocaldarius. J Biol Chem 272:8417-26.

17. Gomes, C. M., T. M. Bandeiras, and M. Teixeira. 2001. A new type-II NADH dehydrogenase from the archaeon Acidianus ambivalens: characterization and in vitro reconstitution of the respiratory chain. J Bioenerg Biomembr 33:1-8.

18. Hamann, N., G. J. Mander, J. E. Shokes, R. A. Scott, M. Bennati, and R. Hedderich. 2007. A cysteine-rich CCG domain contains a novel [4Fe-4S] cluster binding motif as deduced from studies with subunit B of heterodisulfide reductase from Methanothermobacter marburgensis. Biochemistry 46:12875-85.

19. Hedderich, R., J. Koch, D. Linder, and R. K. Thauer. 1994. The heterodisulfide reductase from Methanobacterium thermoautotrophicum contains sequence motifs characteristic of pyridine-nucleotide-dependent thioredoxin reductases. Eur J Biochem 225:253-61.

20. Heinzinger, N. K., S. Y. Fujimoto, M. A. Clark, M. S. Moreno, and E. L. Barrett. 1995. Sequence analysis of the phs operon in Salmonella typhimurium and the contribution of thiosulfate reduction to anaerobic energy metabolism. J Bacteriol 177:2813-20.

21. Hettmann, T., C. L. Schmidt, S. Anemuller, U. Zahringer, H. Moll, A. Petersen, and G. Schafer. 1998. Cytochrome b558/566 from the archaeon Sulfolobus acidocaldarius. A novel highly glycosylated, membrane-bound b-type hemoprotein. J Biol Chem 273:12032-40.

22. Hiller, A., T. Henninger, G. Schafer, and C. L. Schmidt. 2003. New genes encoding subunits of a cytochrome bc1-analogous complex in the respiratory chain of the hyperthermoacidophilic crenarchaeon Sulfolobus acidocaldarius. J Bioenerg Biomembr 35:121-31.

23. Holmes, D. S., and V. Bonnefoy. 2007. Genetic and Bioinformatic Insights into Iron and Sulfur Oxidation Mechanisms of Bioleaching Organisms. In D. E. Rawlings and D. B. Johnson (ed.), Biomining. Springer-Verlag, Berlin Heidelberg.

24. Iwasaki, T., A. Kounosu, M. Aoshima, D. Ohmori, T. Imai, A. Urushiyama, N. J. Cosper, and R. A. Scott. 2002. Novel [2Fe-2S]-type redox center C in SdhC of

archaeal respiratory complex II from Sulfolobus tokodaii strain 7. J Biol Chem 277:39642-8.

25. Jiao, Y., and D. K. Newman. 2007. The pio operon is essential for phototrophic Fe(II) oxidation in Rhodopseudomonas palustris TIE-1. J Bacteriol 189:1765-73.

26. Kanao, T., K. Kamimura, and T. Sugio. 2007. Identification of a gene encoding a tetrathionate hydrolase in Acidithiobacillus ferrooxidans. J Biotechnol 132:16-22.

27. Kappler, U., L. I. Sly, and A. G. McEwan. 2005. Respiratory gene clusters of Metallosphaera sedula - differential expression and transcriptional organization. Microbiology 151:35-43.

28. Kletzin, A. 2007. Metabolism of inorganic sulfur compounds in Archaea, p. 261-276. In R. A. Garrett and H. P. Klenk (ed.), Archaea: Evolution, Physiology, and Molecular Biology. Blackwell.

29. Komorowski, L., W. Verheyen, and G. Schafer. 2002. The archaeal respiratory supercomplex SoxM from S. acidocaldarius combines features of quinole and cytochrome c oxidases. Biol Chem 383:1791-9.

30. Krafft, T., M. Bokranz, O. Klimmek, I. Schroder, F. Fahrenholz, E. Kojro, and A. Kroger. 1992. Cloning and nucleotide sequence of the psrA gene of Wolinella succinogenes polysulphide reductase. Eur J Biochem 206:503-10.

31. Laska, S., F. Lottspeich, and A. Kletzin. 2003. Membrane-bound hydrogenase and sulfur reductase of the hyperthermophilic and acidophilic archaeon Acidianus ambivalens. Microbiology 149:2357-71.

32. Lemos, R. S., C. M. Gomes, and M. Teixeira. 2001. Acidianus ambivalens Complex II typifies a novel family of succinate dehydrogenases. Biochem Biophys Res Commun 281:141-50.

33. Lubben, M., S. Arnaud, J. Castresana, A. Warne, S. P. Albracht, and M. Saraste. 1994. A second terminal oxidase in Sulfolobus acidocaldarius. Eur J Biochem 224:151-9.

34. Lubben, M., B. Kolmerer, and M. Saraste. 1992. An archaebacterial terminal oxidase combines core structures of two mitochondrial respiratory complexes. EMBO J 11:805-12.

35. Lubben, M., A. Warne, S. P. Albracht, and M. Saraste. 1994. The purified SoxABCD quinol oxidase complex of Sulfolobus acidocaldarius contains a novel haem. Mol Microbiol 13:327-35.

36. McDevitt, C. A., G. R. Hanson, C. J. Noble, M. R. Cheesman, and A. G. McEwan. 2002. Characterization of the redox centers in dimethyl sulfide dehydrogenase from Rhodovulum sulfidophilum. Biochemistry 41:15234-44.

37. Muller, F. H., T. M. Bandeiras, T. Urich, M. Teixeira, C. M. Gomes, and A. Kletzin. 2004. Coupling of the pathway of sulphur oxidation to dioxygen reduction: characterization of a novel membrane-bound thiosulphate:quinone oxidoreductase. Mol Microbiol 53:1147-60.

38. Ouchane, S., W. Nitschke, P. Bianco, A. Vermeglio, and C. Astier. 2005. Multiple Rieske genes in prokaryotes: exchangeable Rieske subunits in the cytochrome bc- complex of Rubrivivax gelatinosus. Mol Microbiol 57:261-75.

39. Patridge, E. V., and J. G. Ferry. 2006. WrbA from Escherichia coli and Archaeoglobus fulgidus is an NAD(P)H:quinone oxidoreductase. J Bacteriol 188:3498-506.

40. Pereira, M. M., T. M. Bandeiras, A. S. Fernandes, R. S. Lemos, A. M. Melo, and M. Teixeira. 2004. Respiratory chains from aerobic thermophilic prokaryotes. J Bioenerg Biomembr 36:93-105.

41. Pott, A. S., and C. Dahl. 1998. Sirohaem sulfite reductase and other proteins encoded by genes at the dsr locus of Chromatium vinosum are involved in the oxidation of intracellular sulfur. Microbiology 144 ( Pt 7):1881-94.

42. Purschke, W. G., C. L. Schmidt, A. Petersen, and G. Schafer. 1997. The terminal quinol oxidase of the hyperthermophilic archaeon Acidianus ambivalens exhibits a novel subunit structure and gene organization. J Bacteriol 179:1344-53.

43. Quatrini, R., C. Appia-Ayme, Y. Denis, J. Ratouchniak, F. A. Veloso, J. Valdes, C. Lefimil, S. Silver, F. Roberto, O. Orellana, F. Denizot, E. Jedlicki, D. S. Holmes, and V. Bonnefoy. 2006. Insights into the iron and sulfur energetic metabolism of Acidithiobacillus ferrooxidans by microarray transcriptome profiling. Hydrometallurgy 83:263-272.

44. Rawlings, D. E., and D. B. Johnson. 2007. The microbiology of biomining: development and optimization of mineral-oxidizing microbial consortia. Microbiology 153:315-24.

45. Rouillard, J. M., M. Zuker, and E. Gulari. 2003. OligoArray 2.0: design of oligonucleotide probes for DNA microarrays using a thermodynamic approach. Nucleic Acids Res 31:3057-62.

46. Rzhepishevska, O. I., J. Valdes, L. Marcinkeviciene, C. A. Gallardo, R. Meskys, V. Bonnefoy, D. S. Holmes, and M. Dopson. 2007. Regulation of a Novel Acidithiobacillus caldus Gene Cluster Involved in Reduced Inorganic Sulfur Compound Metabolism. Appl Environ Microbiol 73:7367-72.

47. Schoepp-Cothenet, B., M. Schutz, F. Baymann, M. Brugna, W. Nitschke, H. Myllykallio, and C. Schmidt. 2001. The membrane-extrinsic domain of cytochrome b(558/566) from the archaeon Sulfolobus acidocaldarius performs pivoting movements with respect to the membrane surface. FEBS Lett 487:372-6.

48. Singer, S. W., C. S. Chan, A. Zemla, N. C. VerBerkmoes, M. Hwang, R. L. Hettich, J. F. Banfield, and M. P. Thelen. 2008. Characterization of cytochrome 579, an unusual cytochrome isolated from an iron-oxidizing microbial community. Appl Environ Microbiol 74:4454-62.

49. Stojanowic, A., G. J. Mander, E. C. Duin, and R. Hedderich. 2003. Physiological role of the F420-non-reducing hydrogenase (Mvh) from Methanothermobacter marburgensis. Arch Microbiol 180:194-203.

50. Thorell, H. D., K. Stenklo, J. Karlsson, and T. Nilsson. 2003. A gene cluster for chlorate metabolism in Ideonella dechloratans. Appl Environ Microbiol 69:5585-92.

51. Yarzabal, A., C. Appia-Ayme, J. Ratouchniak, and V. Bonnefoy. 2004. Regulation of the expression of the Acidithiobacillus ferrooxidans rus operon

encoding two cytochromes c, a cytochrome oxidase and rusticyanin. Microbiology 150:2113-23.

52. Yarzabal, A., G. Brasseur, J. Ratouchniak, K. Lund, D. Lemesle-Meunier, J. A. DeMoss, and V. Bonnefoy. 2002. The high-molecular-weight cytochrome c Cyc2 of Acidithiobacillus ferrooxidans is an outer membrane protein. J Bacteriol 184:313-7.

53. Zeng, J., M. Geng, Y. Liu, L. Xia, J. Liu, and G. Qiu. 2007. The sulfhydryl group of Cys138 of rusticyanin from Acidithiobacillus ferrooxidans is crucial for copper binding. Biochim Biophys Acta 1774:519-25.

Table 3.1: Components of respiratory ETC in the order Sulfolobales Homolog present Component Msed ORFs in Reference Proposed function A. ambivalens (17) NDH-II Msed2059 S. metallicus (3) transfer of e- from NADH to quinones A. ambivalens (32) SdhABCD Msed0674-0677 S. tokodaii (24) transfer of e- from succinate to quinones Terminal oxidases: SoxABCDL Msed0285-0291 S. acidocaldarius (16) aa3 –type transferring electrons to O2 (proton pump) SoxEFGHIM Msed0319-0324 S. acidocaldarius (29) bb3 –type transferring electrons to O2 (proton pump) DoxBCE Msed2030-2032 A. ambivalens (14) aa3 –type transferring electrons to O2 (proton pump) SoxNL-CbsAB Msed0500-0504 S. acidocaldarius (22) electron transfer –direction unknown FoxABCD Msed0477-0478, S. metallicus / (5) electron transfer –direction unknown 0480, 0484-0485 S. tokodaii DoxDA Msed0363-0364 A. ambivalens (37) transfer of e- from thiosulfate to quinones A. ferrooxidans (26) TetH Msed0804 A. caldus (9) disproportionation of tetrathionate

98 Table 3.2: M. sedula genome loci responding to ferrous iron and reduced inorganic sulfur compounds (RISC). Y= yeast extract, YFS = Y + ferrous sulfate, YKS = Y + potassium sulfate, YKT = Y + potassium tetrathionate, YS = Y + elemental sulfur. Bonferroni correction for this data set was 5.4. Contrast Fold Cluster ORF change [-log10(p-val)] Details 0289 13.9 16.4 0290 2.4 4.2 soxABCDD’L 0291 YKT vs. YFS 3.3 4.3 Table S7 0285 14.8 17.9 0286 4.9 12.1 0288 2.7 3.1 0323 1.2 0.7 0322 1.5 1.4 soxEFGHIM 0321 YFS vs. YS 3.0 10.5 Table S8 0320 1.6 9.1 0319 1.1 0.6 0324 1.8↓ 2.2 0484 2.0↓ 3.7 0485 14.9 10.6 0480 2.1 3.5 0478 1.5 1.1 foxAA’BCDEFGHIJ 0477 Y vs. YFS 1.0 0.0 Table S8 0475 1.2↓ 0.8 0474 1.4↓ 1.6 0469 1.3↓ 3.0 0468 1.5↓ 8.8 0473 1.6↓ 7.4 0500 5.1 13.3 0501 1.2 0.6 0502 1.7 6.7 soxNL-cbsABA (1147) YFS vs. YKT Table S8 0502 2.6 7.1 (1148) 0503 2.0 7.8 0504 6.4 11.3 0804 31.9 20.0 0812 13.5 13.3 tetH, 0814 8.2 12.1 sre reductase-like / 0815 YKT vs. Y 9.9 16.3 Tables S1,S3 dms reductase-like 0816 3.4 12.8 0817 2.6 8.4 0818 1.6 2.9 1542 5.5 7.0 1543 12.6 11.1 1544 5.5 6.9 1545 6.5 5.1 hdrABC-like 1546 YKT vs. YFS 5.0 5.9 Table S5 1547 1.8 4.4 1548 10.1 12.1 1549 19.0 10.9 1550 12.3 10.0 2030 1.5↓ 2.0 doxBCE 2031 YS vs. YFS 2.4 3.8 Tables S7,S8 2032 1.8↓ 3.4

Figure 3.1: Composite model of respiratory electron transport chain components in the Sulfolobales. Inset table notes components identified in selected Sulfolobales. Sso = S. solfataricus, Sto = S. tokodaii, Sac = S. acidocaldarius, Mse = M. sedula, Aam = A. ambivalens, Sme = S. metallicus. 1Genome sequences are not yet available for these organisms.

100

Figure 3.2: Proposed model of M. sedula respiratory electron transport chain(s) and transcriptional response to tested conditions. Model proposed is based on previous work done in other Sulfolobales. Conditions tested: Y= yeast extract, YFS = Y + ferrous sulfate, YKS = Y + potassium sulfate, YKT = Y + potassium tetrathionate, YS = Y + elemental sulfur. Grayscale patterns represent a differential transcription (minimum log2 fold = 0.9 and –log10(p-value) ≥ 2.5) between specified test conditions, while heat plots depict normalized transcription levels (Lsm = least squares mean) for each test condition compared to an average Lsm = 0. Heat plots were constructed using AFM 4.0 (7). *For Msed2031-2, specified differential transcription was observed for YS vs YFS but not for YKT vs YFS; for Msed0817, specified differential transcription was observed for YKT vs YFS but not for YS vs YFS. Differential transcription log2 fold changes and significance values for each ORF under all test conditions can be found in GEO accession # GSE12044.

101

Chapter 3 Appendix

Components of electron transport chains in the extremely thermoacidophilic crenarchaeon Metallosphaera sedula identified through iron and sulfur compound oxidation transcriptomes

102

Table 3.A1:ORFs implicated in RISC processing Log2 fold change Log2 fold change Log2 fold change Log2 fold change Msed GenBank Annotation for (YFS)-(YS) for (YFS) - (YKT) for (Y)-(YS) for (Y)-(YKT) Proposed ORF (09Apr2008) [-log10(p-value)] [-log10(pvalue)] [-log10(pvalue)] [-log10(p-value)] Annotation 0363 TQO small subunit DoxD 0.1 [0.6] 0.1 [0.3] -0.4 [3.6] -0.4 [3.5] DoxD TQO small subunit DoxA 0364 domain protein 0.2 [1.0] 0.0 [0.1] -0.3 [2.1] -0.4 [3.8] DoxA 0374 TQO small subunit DoxD -0.4 [0.9] 0.1 [0.2] -0.3 [0.9] 0.1 [0.3] DoxD-like putative tetrathionate hydrolase 0804 Pyrrolo-quinoline quinone -3.0 [12.5] -4.9 [19.7] -3.0 [14.8] -5.0 [20.0] (TetH)

Table 3.A2: Blue copper protein differential transcription Log2 fold change Log2 fold change Log2 fold change Log2 fold change Msed GenBank Annotation for (YFS)-(YS) for (YFS)-(YKT) for (Y)-(YFS) for (Y)-(YFS) + Proposed ORF (01May2008) [-log10(p-value)] [-log10(p-value)] [-log10(p-value)] [-log10(p-value)] Annotation sulfocyanin 0323 sulfocyanin 0.3 [0.7] 0.7 [2.3] 0.2 [0.5] 0.3 [0.6] (SoxE) putative 0826 hypothetical protein 0.5 [5.0] 0.3 [2.0] -0.4 [4.4] -0.2 [0.4] sulfocyanin blue (type 1) copper putative 0966 domain protein 0.4 [3.7] 0.2 [1.2] -0.4 [4.1] -0.1 [0.2] rusticyanin blue (type 1) copper putative 1206 domain protein 0.5 [3.2] 0.2 [1.1] -0.2 [1.5] -0.8 [4.4] rusticyanin +Data taken from M. sedula dye-flip partially presented in {Auernik et al, 2008} (GEO Series accession # GSE11296).

103 Table 3.A3: RISC ETC differential transcription (Msed 0810 – 0818) Log2 fold Log2 fold Log2 fold Log2 fold change for change for change for change for Msed GenBank Annotation (YFS)-(YS) (YFS)-(YKT) (Y)-(YS) (Y)-(YKT) Proposed ORF (13Mar2008) [-log10(p-value)] [-log10(p-value)] [-log10(p-value)] [-log10(p-value)] Annotation regulatory 0810 Hypothetical protein -1.1 [3.3] -2.1 [7.8] -0.3 [0.6] -1.3 [4.0] domain 0811 Hypothetical protein -0.3 [1.0] 0.1 [0.2] -0.2 [0.8] 0.2 [0.4] GTP-binding Psr/Dms/Ph Nitrite reductase s-like membrane component- membrane 0812 like protein -2.0 [6.3] -4.2 [14.7] -1.6 [5.8] -3.8 [13.3] anchor TPR involved in TPR-repeat containing multi-subunit 0813 protein -0.4 [2.4] -0.3 [1.5] -0.1 [0.5] -0.0 [0.0] assembly Anaerobic DmsA/PsrA/ 0814 dehydrogenase -2.2 [8.4] -2.9 [11.4] -2.4 [11.1] -3.0 [12.1] PhsA-like DmsB/SreB/ 4Fe-4S ferredoxin, FeS PsrB/PhsB- 0815 binding domain protein -2.0 [9.5] -3.2 [15.8] -2.1 [12.3] -3.3 [16.3] like DmsD/SreE/ ClrD/TorD- 0816 Hypothetical protein -1.3 [8.9] -1.7 [12.4] -1.3 [9.3] -1.8 [12.8] like SreD-like with weak NADH:quino ne (subunit 4Fe-4S ferredoxin, FeS 1) oxidored 0817 binding domain protein -0.6 [2.7] -1.3 [7.5] -0.7 [4.6] -1.4 [8.4] CTD 0818 Hypothetical protein -0.4 [1.1] -0.9 [4.2] -0.2 [0.4] -0.7 [2.9] hypothetical

104 Table 3.A4: Homologs to RISC-induced ORFs (Msed 0810 - 0818) M. sedula ORF# 0810 0811 0812 0813 0814 0815 0816 0817 0818 Homolog (% Identity) Sulfolobus 2798 2799 2797 2796 2795 2794 2793 2792 2791 solfataricus (63) (49) (75) (62) (80) (88) (64) (70) (50) Sulfolobus 1843 - 1842 1841 1840 1839 1838 1837 1836 tokodaii (35) (62) (43) (83) (90) (50) (50) (45) Sulfolobus 2103 2104 2109 2105 2102 2101 2100 2106 2107 acidocaldarius (39) (19) (56) (40) (73) (81) (46) (45) (34) Acidianus SreC SreA SreB SreE SreD ambivalens1 (-) (24) (28) (24) (22) Acidithiobacillus - - 0918 - 0920 0919 - - - ferrooxidans (22) (29) (28) Samonella PhsC PhsA PhsB typhimurium2 (-) (22) (34) Wolinella PsrC PsrA PsrB succinogenes3 (26) (25) (32) Ideonella ClrC ClrA ClrB ClrD dechloratans4 (-) (37) (28) (25) Rhodovulum DdhC DdhA DdhB DdhD sulfidophilum5 (-) (23) (29) (24)

1Aam SreABCDE (AJ345004) {Laska et al, 2003 } was used as a query against Mse genome. 2Salty PhsABC (L32188) {Heinzinger et al, 1995} was used as a query against Mse genome. 3Wsu PsrABC (X65042){Krafft et al, 1992} was used as a query against Mse genome. 4Ide ClrABDC (AJ566363){Thorell et al, 2003} was used as a query against Mse genome. 5Rsu DdhABDC (AF453479){McDevitt et al, 2002} was used as a query against Mse genome.

105

Table 3.A5: RISC ETC differential transcription (Msed 1542 - 1550) Log2 fold Log2 fold Log2 fold Log2 fold change for change for change for change for Msed GenBank Annotation (YFS)-(YS) (YFS)-(YKT) (Y)-(YS) (Y)-(YKT) Proposed ORF (13Mar2008) [-log10(p-value)] [-log10(p-value)] [-log10(p-value)] [-log10(p-value)] Annotation CoB-CoM heterodisulfide 1542 reductase -2.1 [5.5] -2.5 [7.0] -2.0 [6.8] -2.4 [6.8] HdrB1 Heterodisulfide reductase 1543 HdrC-like protein -3.5 [10.7] -3.7 [11.1] -2.5 [9.0] -2.7 [7.6] HdrC1 Hdr-associated 1544 Hypothetical protein -2.2 [6.0] -2.5 [6.9] -1.7 [5.5] -2.0 [5.1] protein FAD-dependent pyridine nucleotide-disulphide 1545 oxidoreductase -2.5 [4.6] -2.7 [5.1] -1.9 [3.5] -2.1 [3.5] HdrA 1546 Cys-rich domain (DUF224) -2.2 [5.3] -2.3 [5.9] -1.6 [4.4] -1.8 [4.0] HdrB2 HdrC2; High lsms Heterodisulfide reductase under all 1547 HdrC-like protein 0.1 [0.3] -0.9 [4.4] 0.3 [1.4] -0.7 [3.0] conditions tested hypothetical 1548 SirA family protein -3.0 [10.9] -3.3 [12.1] -1.3 [4.8] -1.6 [4.9] regulatory protein Hdr-associated 1549 Hypothetical protein -2.8 [6.7] -4.2 [10.9] -1.1 [2.1] -2.5 [5.8] protein Hdr-associated 1550 DsrE family protein -2.3 [5.6] -3.6 [10.0] -0.8 [1.4] -2.2 [5.1] protein

106 Table 3.A6: Sequences similar to RISC-induced ORFs (Msed 1542 - 1550) M. sedula ORF# 1542 1543 1544 1545 1546 1547 1548 1549 1550 Homolog (% Identity) Sulfolobus 1135 1134 1133 1131 1129 1127 7239 1126 1125 solfataricus (80) (83) (54) (84) (90) (83) (87) (81) (92) Sulfolobus 1870 1871 1872 1873 1874 1875 203 1876 1877 tokodaii (85) (84) (62) (86) (88) (82) (92) (80) (88) Sulfolobus 0325 0326 0327 0328 0329 0334 0335 0336 0337 acidocaldarius (78) (76) (54) (83) (85) (84) (86) (79) (88) Acidithiobacillus 0552 0551 0550 0549 0548 0547 0545 0546 0554 ferrooxidans (39) (34) (26) (40) (50) (43) (37) (36) (28) Aquifex 0400 0398 0397 0395 0392 0391 1421 0389 0390 aeolicus (44) (38) (25) (40) (59) (43) (35) (30) (29) 0394 (44) Methanococcus 0863 0864 - 1190 0743 0744 0990 - 0760 jannaschii (30) (33)* (38)* (24) (30)* (35) (28) Methanopyrus 0572 0573 - 0265 0572 0573 0257 - 0008 kandleri (30) (30)* (36)* (26) (27)* (43) (23) Methanothermobacter 1879 1878 - 1381 1879 1878 - - - thermoautotrophicus (29) (32)* (39)* (25) (26)* Methanosarcina 4237 4236 - 2868 4237 4236 0810 0809 - acetivorans (28) (28)* (38) (22) (30)* (36) (33) Methanosarcina 0980 0979 - 0056 0980 0979 1957 1956 - mazei (30) (28)* (36) (22) (27)* (32) (33)

*similarity for the central region (100 of 230aa/240 out of 650-800aa for HdrA-likes) of the sequence only

107 Table 3.A7: Terminal oxidases potentially involved in RISC oxidation Log2 fold Log2 fold Log2 fold Log2 fold change for change for change change for Msed GenBank Annotation (YFS)-(YS) (YFS)-(YKT) for (Y)-(YS) (Y)-(YKT) Proposed ORF (09Apr2008) [-log10(p-value)] [-log10(p-value)] [-log10(p-value)] [-log10(p value)] Annotation Conserved 0283 Hypothetical protein -0.9 [4.9] -0.5 [2.1] -0.9 [5.9] -0.6 [2.6] hypothetical Conserved 0284 Hypothetical protein -2.0 [8.3] -1.8 [7.0] -1.1 [3.9] -0.8 [2.3] hypothetical 0285 Hypothetical protein -2.4 [11.2] -3.9 [17.9] -0.2 [0.5] -1.7 [7.6] SoxD 0286 Hypothetical protein -1.5 [7.2] -2.3 [12.1] 0.2 [0.6] -0.6 [2.0] SoxD’ Rieske (2Fe-2S) domain 0288 protein -1.2 [2.4] -1.4 [3.1] -0.5 [0.9] -0.7 [1.1] SoxL (Rieske) cytochrome c oxidase, 0289 subunit II -3.2 [13.8] -3.8 [16.4] -1.7 [8.5] -2.4 [10.1] SoxA (coxII) 0290 Cytochrome-c oxidase -1.2 [3.9] -1.3 [4.2] -1.0 [3.7] -1.1 [3.2] SoxB (coxI) Cytochrome b/b6, N- 0291 terminal domain -1.3 [2.8] -1.7 [4.3] -0.9 [2.1] -1.3 [3.0] SoxC (cyt b) protein of unknown function Conserved 0292 DUF1404 -0.2 [1.2] -0.5 [4.4] -0.3 [2.2] -0.6 [5.9] membrane protein 2030 Hypothetical protein 0.6 [2.0] 0.5 [1.7] 0.5 [2.0] 0.4 [1.2] DoxE 2031 hypothetical protein -1.3 [3.8] -0.7 [1.5] -0.5 [1.0] 0.1 [0.2] DoxC (coxII) cytochrome c oxidase, 2032 subunit I 0.8 [3.4] 0.5 [1.5] -0.1 [0.4] -0.5 [1.7] DoxB (coxI) 0570 Cytochrome-c oxidase -1.0 [3.7] -1.1 [4.0] -1.1 [5.7] -1.2 [4.7] DoxB-like (coxI)

108

Table 3.A8: FOX ETC differential transcription

Log2 fold change Log2 fold change Log2 fold change Log2 fold change for for (YE)-(YEF) Msed for (YFS)-(YS) for (YFS)-(YKT) [- (Y)-(YFS) [-log10(p-value)] Proposed ORF GenBank Annotation (02May2008) [-log10(p-value)] log10(p-value)] [-log10(p-value)] Annotation 0319 hypothetical protein 0.1 [0.6] 0.6 [6.3] -0.3 [2.9] 0.1 [0.5] soxI cytochrome c oxidase, cytochrome oxidase, 0320 subunit II 0.7 [9.1] 0.3 [3.3] -0.5 [7.9] -0.1 [0.3] subunit II (soxH) Cytochrome b/b6, 0321 N-terminal domain 1.6 [10.5] 1.2 [7.9] -1.3 [10.2] -0.5 [1.5] cytochrome b (soxG) 0322 Rieske (2Fe-2S) domain protein 0.6 [1.4] 0.3 [0.6] -0.2 [0.5] 0.1 [0.1] Rieske protein (soxF) 0323 Sulfocyanin 0.3 [0.7] 0.7 [2.3] 0.2 [0.5] 0.3 [0.6] Sulfocyanin (soxE) Cyctochrome oxidase, 0324 Cytochrome-c oxidase -0.8 [2.2] -0.2 [0.4] 1.1 [3.9] 0.4 [0.9] subunit I/III (soxM) 0467 major facilitator superfamily 0.7 [5.9] 0.3 [1.8] -0.6 [5.8] -1.5 [8.0] putative signal-transduction protein 0468 with CBS domains 0.5 [5.6] 0.2 [1.5] -0.6 [8.8] -0.0 [0.1] foxH 0469 0469 0.4 [1.8] 0.3 [1.6] -0.4 [3.0] -0.4 [2.0] (2o) hypothetical protein 0.4 [2.8] 0.2 [1.1] -0.3 [2.6] 0.1 [0.2] foxG 0473 hypothetical protein 1.1 [10.0] 0.3 [1.8] -0.7 [7.4] -0.2 [1.5] foxJ 0474 hypothetical protein 0.4 [1.0] 0.4 [1.4] -0.4 [1.6] -0.3 [0.6] foxF (NuoN/ML-like) 0475 hypothetical protein 1.1 [5.0] 0.3 [0.6] -0.3 [0.8] 0.0 [0] foxE

cytochrome b558-566 0477 hypothetical protein 0.3 [1.2] 0.6 [3.2] 0.0 [0.0] 0.8 [2.6] (cbsB/foxD)

cytochrome b558-566 0478 hypothetical protein -0.2 [0.2] 1.0 [2.0] 0.6 [1.1] 1.7 [7.3] (cbsA/foxC) 0479 hypothetical protein 0.3 [0.3] 1.5 [3.2] 0.5 [0.8] 2.4 [8.1] cytochrome c oxidase, cytochrome oxidase, 0480 subunit II -0.4 [0.7] 0.5 [0.9] 1.1 [3.5] 1.9 [10.5] subunit II (soxH/foxB) 0482 hypothetical protein -1.0 [3.4] -1.1 [4.0] 1.9 [9.7] 1.1 [3.5] 0483 hypothetical protein -0.5 [2.0] -0.6 [2.6] 0.6 [3.1] 0.0 [0.1] foxI cytochrome oxidase, cytochrome c oxidase, subunit I 0484 subunit I 0.9 [2.5] 1.4 [4.9] -1.0 [3.7] -0.9 [4.5] (soxB2/foxA1)

109 (Table 3.A8 continued) cytochrome cytochrome c oxidase, oxidase, subunit I 0485 subunit I -3.1 [6.4] -2.5 [4.8] 3.9 [10.6] 0.7 [1.7] (soxB3/foxA2) FAD-dependent pyridine nucleotide-disulphide 0487 oxidoreductase 0.4 [5.1] 0.4 [4.4] -0.3 [2.7] 0.1 [0.1] DNA polymerase, beta domain 0489 protein region 2.2 [21.1] 1.2 [13.0] -1.6 [19.4] -0.5 [4.7] Cytochrome b/b6, cytochrome b 0500 N-terminal domain 2.1 [11.6] 2.4 [13.3] -1.8 [12.3] -1.6 [4.1] (soxN) Rieske (2Fe-2S) domain 0501 protein -0.5 [1.2] 0.3 [0.6] -0.4 [0.8] -0.9 [3.0] Rieske (soxL) 0502 cytochrome b558- 0502 1.6 [8.8] 1.4 [7.1] -1.2 [7.6] -0.9 [5.5] 566 (cbsA/foxC- (2o) pseudogene 1.1 [9.9] 0.8 [6.7] -0.8 [8.9] -1.4 [4.4] like) cytochrome b558- 566 (cbsB/foxD- 0503 hypothetical protein 0.8 [6.3] 1.0 [7.8] -0.7 [6.6] -0.8 [3.6] like) cytochrome b558- 566 (cbsA/foxC- 0504 hypothetical protein 2.4 [9.7] 2.7 [11.3] -1.6 [7.7] -1.0 [1.7] like) plasma-membrane proton- 0505 efflux P-type ATPase 0.4 [4.0] 0.6 [5.8] -0.6 [7.6] 0.0 [0.0] 2030 hypothetical protein 0.6 [2.0] 0.5 [1.7] -0.1 [0.2] -0.3 [1.2] doxE Cytochrome oxidase subunit II 2031 hypothetical protein -1.3 [3.8] -0.7 [1.5] 0.8 [2.4] -0.1 [0.4] (doxC) Cytochrome cytochrome c oxidase, oxidase subunit I 2032 subunit I 0.8 [3.4] 0.5 [1.5] -1.0 [5.6] -0.6 [7.9] (doxB)

110 Table 3.A9: ORFs with significant differential transcription

Msed GenBank Annotation Log2 fold [-log10(p- Notes/ ORF Contrast change value)] Comments YFS vs YS -1.1 2.6 YFS vs YKT -2.9 9.3 Y vs YS -1.4 4.5 0371 hypothetical protein Y vs YKT -3.2 10.4 RISC-induced YFS vs YS -1.7 5.2 YFS vs YKT -3.3 12.1 protein of unknown function Y vs YS -1.7 6.4 0372 DUF395, YeeE/YedE Y vs YKT -3.4 12.4 RISC-induced 2— YFS vs YKT -3.5 10.8 S4O6 induced; 0373 SirA family protein Y vs YKT -3.5 10.7 similar to 1548 regulator YFS vs. YS 1.0 6.8 pyruvate flavodoxin /ferredoxin YFS vs. YKT 0.7 4.0 Fe(II)-induced 0509 oxidoreductase domain protein YFS vs. Y 0.8 4.9 YFS vs. YS 1.0 8.2 YFS vs. YKT 0.8 6.2 Fe(II)-induced 0511 Radical SAM domain protein YFS vs. Y 0.9 7.7 YFS vs. YS 1.5 12.2 YFS vs. YKT 1.5 11.8 Fe(II)-induced 0512 Radical SAM domain protein YFS vs. Y 1.4 11.1 YFS vs. YS 1.4 7.8 YFS vs. YKT 1.7 9.3 Fe(II)-induced 0513 β-lactamase domain protein YFS vs. Y 1.5 10.1 YFS vs. YS 1.1 10.4 YFS vs. YKT 1.1 10.5 Fe(II)-induced 0514 peptidase U32 YFS vs. Y 1.0 10.8 YFS vs. YS 1.8 13.4 YFS vs. YKT 1.3 10.0 Fe(II)-induced 0515 Radical SAM domain protein YFS vs. Y 1.9 15.3 YFS vs. YS 2.0 14.1 YFS vs. YKT 1.7 11.8 Fe(II)-induced 0518 hypothetical protein YFS vs. Y 2.1 17.5 YFS vs YS -3.8 15.1 YFS vs YKT -2.6 10.5 Y vs YS -3.7 17.4 RISC-induced; 1905 hypothetical protein Y vs YKT -2.5 10.0 membrane- associated

111

Figure 3.A1: Normalized transcription levels (Lsms) of M. sedula blue copper proteins for all conditions tested. Average normalized transcription level is Lsm = 0.

Figure 3.A2: Ratio of foxA/foxA’ transcription levels (Lsm_Msed0484/Lsm_Msed0485) for conditions tested.

112

Figure 3.A3: Phylogram (constructed using ClustalW2) of DMSO reductase-like sequences (α subunits) labeled with GenBank identifiers.

Figure 3.A4: Phylogram (constructed using ClustalW2) of heterodisulfide reductase-like sequences (HdrB homologs) listed with GenBank identifier

113

Chapter 4

Physiological versatility of the extremely thermoacidophilic archaeon Metallosphaera sedula supported by heterotrophy, autotrophy, and mixotrophy transcriptomes

Kathryne S. Auernik and Robert M. Kelly

Department of Chemical and Biomolecular Engineering North Carolina State University Raleigh, NC 27695-7905

submitted to Journal of Bacteriology (March, 2009)

114

ABSTRACT

The versatile growth physiology of the extremely thermoacidophilic crenarchaeon

Metallosphaera sedula was examined in conjunction with whole genome transcriptional response analysis. Growth rates characterized by doubling times under mixotrophic conditions (4 hour doubling time on tryptone, H2, CO2) were observed to be faster than growth rates under autotrophic (12 hour doubling time on H2, CO2) or heterotrophic (5 hour doubling time on tryptone) conditions. Heterotrophic and autotrophic transcriptomes revealed central carbon metabolism function consistent with other Sulfolobales, upregulation of the 3-hydroxpropionate/4-hydroxybutyrate cycle enzymes identified in M. sedula, and ruled in and out specific candidate ORFs for the remaining uncharacterized enzymes of this inorganic carbon-fixing cycle (i.e. 4-hydroxybutyryl-CoA dehydratase, crotonyl-CoA hydratase, 3-hydroxybutyryl-CoA dehydrogenase, and acetoacetyl-CoA β- ketothiolase). The primary hydrogenase responsible for H2 utilization as an inorganic energy source could also be identified (Msed0944-Msed0946) and possible links to ATP and/or reducing power generation are discussed. Mixotrophic transcriptomes showed a strong preference for organic carbon, with concomitant use of H2 and organic carbon as energy sources, implying that faster growth rates observed with mixotrophy were likely the result of relief of a bioenergetic limitation present during heterotrophic growth.

115 INTRODUCTION

Microbial mixotrophy has been defined as: 1) the capacity to assimilate both organic and inorganic carbon sources simultaneously (30, 34), or 2) the utilization of an inorganic energy source in combination with an organic carbon source (1, 23, 35). Common to either concept is the idea that mixotrophy involves some blend of heterotrophic and autotrophic traits in the same microorganism. Whether mixotrophy is an environmental adaptation or an essential physiological characteristic arising from an evolutionary process is not clear, although microorganisms with this feature should be more competitive in dynamic ecosystems. Microbial mixotrophy has mostly been studied as a complement to photosynthetic processes. In land plants, such as green orchids and shrubs (pyroloids), carbon can be acquired both from inorganic photosynthetic sources and from organic fungal sources (30). Plankton from the eukaryotic genus Ochromonas can out-compete their autotrophic (phototrophic) and heterotrophic (phagotrophic) peers by utilizing light as an energy source and preying on food algae and bacteria for organic carbon (35). Metabolic flux balance analysis in the planktonic alga Chlamydomonas reinhardtii growing under mixotrophic conditions suggests that the ratio of inorganic/organic carbon assimilated to biomass is a function of the magnitude of light (or inorganic energy) available. Thus, increased light stimulates the incorporation of organic carbon into biomass (influenced by the relatively low energy content of acetate) (6). Non-photosynthetic mixotrophy has also been observed. In the bacterium Xanthobacter tagetidis, mixotrophic growth occurs in the presence of organic carbon sources (thiophene-2-carboxylic acid and/or acetic acid) and an inorganic energy source (thiosulfate) (23). In the hyperthermophilic archaeon, Pyrobaculum

116 islandicum, mixotrophic growth on organic carbon sources requires the availability of a mixture of (in)organic energy sources (yeast extract, acetate, and H2) (1). In some facultatively chemoautotrophic bacteria, such as Ralstonia or Pseudomonas species, the carbon fixation process may be regulated by the availability of organic carbon, as the Calvin cycle exhibited de-repression facilitating inorganic carbon assimilation when organic carbon sources were limiting (31). In the nitrite-oxidizing bacterial genus Nitrobacter, some species, such as Nitrobacter winogradskyi and Nitrobacter hamburgensis X14, can grow heterotrophically on organic carbon, but cannot use organic carbon for mixotrophic growth unless inorganic carbon is also present (33).

Metallosphaera sedula is an aerobic, extremely thermoacidophilic archaeon that grows heterotrophically on peptides or yeast extract, autotrophically by fixing CO2, using molecular hydrogen as a reductant, and presumably mixotrophically on casamino acids and

FeSO4 or pyrite (1, 2, 11, 13, 17, 26). Among the genome-sequenced extreme thermoacidophiles, M. sedula stands out, given its capacity to oxidize organic carbon, reduced metal species, sulfur, and H2 for energy to support growth (see Table 4.1). In addition to the common Sulfolobales terminal oxidases (SoxABCD and SoxM complexes) which contribute to ATP generation via proton motive force generation, M. sedula also has

ORFs encoding putative enzymes which allow it to derive energy from Fe(II) (FoxABCD,

Rus), and H2 (NiFe hydrogenases), a combination not known be present in any other genome sequenced thermoacidophile. Recent studies of M. sedula’s inorganic carbon fixation cycle (5), in conjunction with genome sequence analysis (3), facilitate analysis of mixotrophic growth characteristics in M. sedula. Here, we examine the physiological

117 versatility of M. sedula, using transcriptional response analysis to explore elements of non- photosynthetic mixotrophy.

MATERIALS AND METHODS

Growth of M. sedula cultures. M. sedula (DSMZ 5348) was grown aerobically at

70°C in an orbital shaking oil bath at 70 rpm on DSMZ 88 medium (pH 2). Three cultivation conditions (each with biological repeats) were followed for growth characteristics: heterotrophic cultures were supplemented with tryptone (0.1% final concentration in medium), autotrophic cultures had a H2+CO2 mixture added to culture bottle headspace

(after first removing an equivalent volume of air) so that initial culture headspace gas contained 7% CO2, 14% O2, 28% H2, with the balance N2, and mixotrophic cultures contained both the heterotrophic supplement as well as the adjusted headspace composition of the autotrophic cultures. Cultures were acclimated to the specific growth conditions through three passages before harvesting. On the fourth passage, cells were inoculated into two 1-liter bottles per culture, containing 300-ml of media (initial densities:

1.0 x 107 cells/mL in both heterotrophic cultures, 7.3 x 106 and 1.1 x 107 cells/mL in autotrophic cultures, and 6.2 x 106 and 6.3x106 cells/mL in mixotrophic cultures). Cell densities were measured using epifluorescence microscopy. Cultures were harvested during exponential phase growth by first rapidly chilling and then centrifuging at 9,510 x g for 15 min at 4°C.

118 M. sedula oligonucleotide microarray construction and transcriptional response analysis. A spotted, whole-genome oligonucleotide microarray, based on at least 2,256 protein-coding open reading frames (ORFs), was constructed (2,328 probes with 5 replicates of each, yielding 11,640 spots per array), as described previously (2, 3).

Probes (60-mers) were designed using OligoArray, version 2.1, software (29) and synthesized by Integrated DNA Technologies (IDT) (Coralville, IA). RNA was extracted

(Trizol; Invitrogen) and purified (RNeasy Mini Kit; Qiagen), reverse transcribed (Superscript

III; Invitrogen), re-purified, labeled with either Cy3 or Cy5 dye (GE Healthcare), and hybridized to one of eight microarray slides (Corning). Slides were scanned on a Packard

BioChip Scanarray 4000 scanner, and raw intensities were quantitated using ScanArray

Express software (version 2.1.8). Normalization of data and statistical analysis were performed using JMP Genomics, version 3.1, software (SAS, Cary, NC). In general, significant differential transcription, or “response,” was defined to be relative changes at or above 2 (i.e., a log2 value of ±1 means a two-fold change) with significance values at or above the Bonferroni correction, which was 5.4 (equivalent to a P value of 4.0 x 10- 6) for this data set. With biological repeats for each condition tested, the 4 possible combinations

(i.e., for autotrophy vs heterotrophy, 4 comparisons A1-H1, A1-H2, A2-H1, and A2-H2) are calculated as the average log2 fold changes and significance values and then reported in the text as the equivalent fold changes and p-values (for readers not used to thinking in terms of log2 fold changes and significance values).

Comparative genomics analysis. For comparative genomics, M. sedula gene sequences and annotations (including signal peptide predictions using SignalP 3.0) were

119 downloaded from the Joint Genome Institute (JGI) microbial genome website

(http://genome.ornl.gov/microbial/msed/28feb07/) or GenBank. M. sedula signal peptide predictions were also checked using PRED-SIGNAL trained on a data set of experimentally verified archaeal signal peptides (4). Other sequences referenced were obtained from the

GenBank. The Basic Local Alignment Search Tool (BLAST) searches were conducted using a BLOSUM62 matrix with either the NCBI protein-protein blast program (BLASTP) against the Swissprot database, the non-redundant database, or against the specific sequenced genomes of interest (in either GenBank or JGI/Oak Ridge National Laboratory databases).

Microarray data accession number. Raw data, as well as final log2 relative changes and significance values (where significance = -log10(p-value)), have been deposited in the NCBI Gene Expression Omnibus (GEO) database under series accession number GSE14978.

RESULTS AND DISCUSSION

Growth physiology. M. sedula culture characteristics varied as a function of growth mode. Figure 4.1 shows growth of M. sedula on the 3rd passage for three growth modes: heterotrophy, autotrophy and mixotrophy. Mixotrophically-grown cells reached stationary phase (approximately 6 x 108 cells/mL) at around 18 hours, with an average doubling time of 3.7 hours. Heterotrophically-grown cells reached stationary phase (approximately 9 x

108 cells/mL) at around 48 hours with an average doubling time of 5.0 hours.

120 Autotrophically grown cells had an average doubling time of 11-13 hours but did not appear to reach stationary phase during the time period monitored (maximum cell density observed approximately 3 x 108 cells/mL). The faster growth rate observed with mixotrophically- grown cells, compared to both autotrophically and heterotrophically grown cells, suggested that both inorganic and organic carbon (and/or energy sources) were being utilized to enhance growth rate.

Heterotrophic growth. At least 20 loci in the genome appeared to respond to heterotrophy, with up-regulation of ORFs under heterotrophic growth compared to autotrophic and/or mixotrophic growth (Table 4.2). A significant portion (~40%) of these

ORFs are annotated as hypothetical proteins. The up-regulation of glutamate dehydrogenase (Msed_2074) on heterotrophy vs. autotrophy is characterisitic of excess nitrogen during growth on peptides and is consistent with predictions for S. solfataricus glutamate dehydrogenase presence on peptide- vs. carbohydrate-supplemented media

(32). Succinyl-CoA synthetase (Msed_1581-Msed_1582), a TCA cycle intermediate (to be discussed below), was transcribed at its highest on heterotrophy. Also up-regulated were

ORFs with non-specific annotations as transporters (Msed_0993, Msed_1095, Msed_1180, and Msed_1772). Of particular interest were: 1) the ORFs clustered with Msed_1180 that are similar to Swissprot database entries for glycerol-3-phosphate import/processing systems (i.e. Msed_1177, Msed_1178, Msed_1179), and 2) the ORFs following

Msed_1772, which appear to be involved in ring cleavage or breakdown of aromatic amino acids (Msed_1774-Msed_1777).

121 Considering the organic carbon source provided was tryptone, ORFs in the genome with annotations specifically related to peptide transport were also examined. Four genome loci regions are annotated as oligo-/dipeptide ABC transporters: Msed_0440-Msed_0447,

Msed_1046-Msed_1052, Msed_1379-Msed_1382, and Msed_2203-Msed_2207 (Table

4.A1). Minimally, all 4 regions appear to contain 2 transmembrane/permease subunits having 5 to 7 predicted transmembrane segments and 2 ATPase subunits. The larger permease subunit in each pair is also predicted to contain a signal peptide (Msed_1048, a smaller permease subunit is also predicted to have a signal peptide). All regions, except for

Msed_1379-Msed_1382, appear to also contain a large (675-872aa) extracellular solute binding protein. Only Msed_0441 contains a predicted C terminal domain (CTD) transmembrane region, which may serve to tether the protein to the membrane. Two loci also contain peptidases (Msed0_440 - S9 family and Msed_1052 - M19 family) and either extra hypothetical proteins with predicted transmembrane regions (Msed_0442,

Msed_0447), or an extra ATPase (Msed_1051). Permease and ATPase components in all

4 loci bear some similarity (20-45%ID) to the peptide transporters (Opp/App) in Bacillus subtilis (19, 27). None of the extracellular solute binding proteins (Msed_0441, Msed_1046,

Msed_2203) share any similarity to the recently characterized protein in archaeon

Aeropyrum pernix (24); however, extracellular proteins in an acidic environment (M. sedula pH 2) may have significantly different aa sequences than their neutral pH equivalents (note that A. pernix grows at pH 5-9). M. sedula extracellular solute binding protein sequences

Msed_1046 and Msed_2203 have homologs in other thermoacidophile genomes

(Sulfolobus solfataricus, Sulfolobus tokodaii, Sulfolobus acidocaldarius, Thermoplasma acidophilum, Thermoplasma volcanium, Picrophilus torridus, and Caldivirga

122 maquilingenesis), while sequences related to Msed0441 only appear in the three sequenced Sulfolobus species.

In general, normalized transcription levels (lsmeans) of these 4 loci were such that

Msed_0440-Msed_0447 > Msed_2203-Msed_2207 > Msed_1046-Msed_1052 >

Msed_1379-Msed_1381. Extracellular solute binding sequences Msed_0441 and

Msed_2203 were consistently transcribed at high levels (lsms 3.7-4.3 and 3.3-3.8, respectively) for all conditions tested. Differential transcription was noted in the

OppCD/AppCD-like subunits (smaller permease subunit and larger ATPase subunit) of 2 transporters (Msed_1048-Msed_1049 and Msed_2205-Msed_2206) with up-regulation under autotrophic conditions relative to heterotrophic conditions (for Msed1048-

Msed_1049: 2.7- fold/3.0-fold with p-values 8.7 x 10-05/5.8 x 10-07) or mixotrophic conditions

(for Msed_2205-Msed_2206: 1.9-fold/3.7-fold with p-values 3.2 x 10-04/1.3 x 10-07). The M19 family peptidase (Msed_1052) was also transcribed at significantly higher levels on mixotrophy compared to autotrophy (2.4-fold with p-value 7.9 x 10-05).

It is not known how M. sedula metabolizes organic carbon, although previous work with Sulfolobus solfataricus (32) focused on enzymes in central carbon metabolism

(glycolysis, gluconeogenesis, and the TCA cycle) as they responded to glucose vs. yeast extract/ tryptone carbon sources. Figure 4.2, based on the closest M. sedula homologs (as determined by BLASTP similarity searches) to the S. solfataricus enzymes, summarizes transcriptional profiles for autotrophic (CO2 carbon source, biological repeats “A1” and “A2”), heterotrophic (tryptone carbon source, biological repeats “H1” and “H2”) and mixotrophic growth (CO2 and tryptone carbon sources, biological repeats “M1” and “M2”). Little or no differential transcription of TCA cycle enzymes was noted, except for the enzymes

123 responsible for forming (2-oxoacid:ferredoxin oxidoreductase, Msed_0524-Msed_0525) and depleting (succinyl-CoA synthetase, Msed_1581-Msed_1582) the succinyl-CoA intermediate, which were transcribed at significantly higher levels on heterotrophy than autotrophy (Table 4.A2). It should be noted that Msed_0525 appears to be a fusion of pyruvate: ferredoxin oxidoreductase (Pfor) γ and α subunits, while Msed_0524 appears to contain the β subunit and a CTD similar to pyruvate dehydrogenase EI component. Within the M. sedula genome, there appears to be 4 additional Pfor-like clusters, including

Msed_0306-Msed_0309 (Pforγδαβ) that is transcribed at consistent levels under all conditions (lsms 2.0 to 2.4), Msed_0507-Msed_0510 (Pforγδαβ) that appears to be up- regulated on autotrophic and mixotrophic conditions compared to heterotrophic,

Msed_1199-Msed_1201 (Pforβαδ) that shows signs of down-regulation on mixotrophy, and

Msed_1596-Msed_1597 (Pforγδα) that was down-regulated on autotrophy (Table 4.A2).

Note that Msed_1597 is predicted to have a signal peptide, and Msed_1598 is annotated as a helix-turn-helix transcriptional regulator with a short region in front of the HTH motif having similarity to a Pfor γ subunit. Transcriptional data suggest that Msed_0507-

Msed_0509 is the most likely candidate to encode an enzyme with pyruvate synthase activity feeding acetyl-CoA into gluconeogenesis (Figure 4.3).

The ORFs encoding the components of gluconeogenesis (see Figure 4.3) were identified by using BLASTP with S. solfataricus query sequences (from (32)) or

Thermococcus kodakarensis query sequences (for the ribulose monophosphate pathway described in (22)) against the M. sedula genome. With a few exceptions, such as the putative pyruvate synthase (Msed_0507-Msed_0509) and phosphoenolpyruvate synthase

(Msed_1694) that were up-regulated on autotrophy compared to heterotrophy, minimal

124 differential transcription was observed in the ORFs comprising gluconeogenesis (Figure

4.3). This was not unexpected, since no carbohydrates were included in the growth media.

Consistent with this, general transcriptional levels of gluconeogenesis appear to be higher than for Entner-Doudoroff glycolysis. A notable exception was a second phosphoglycerate mutase (Msed_0773); this is consistent with the S. solfataricus studies (32) that showed that the second phosphoglycerate mutase (SSO2236) was not detectable by transcriptomics or proteomics, and suggests that the first copy (Msed_2238/SSO0417) is responsible for primary function in gluconeogenesis. The other exception is the aldehyde oxidoreductase (γ/β, Msed_0298-Msed_0299), which is a possible M. sedula homolog to an

S. acidocaldarius complex with oxidoreductase activity on glyceraldehyde in cell extracts

(18). It is possible that other sequences may encode the appropriate activity, or that the enzyme encoded by these sequences may have additional functions (i.e. a branch point for an additional pathway), prompting higher transcriptional levels. Finally of note is the down- regulation of a putative fructose 1,6 bisphosphatase (Msed_2259) and a putative 3- hexulose 6-phosphate synthase (HPS, Msed_0227) under mixotrophic growth conditions.

In T. kodakarensis the ribulose monophosphate pathway substitutes for the pentose phosphate pathway (PPP) in the production of ribulose 5-phosphate (22). If the cycle were to be operating in M. sedula (which lacks annotations for a complete PPP), the fructose-6- phosphate starting substrate could be provided from fructose 1,6 bisphosphatase

(Msed_2259). While biochemical confirmation is needed to determine whether the ribulose monophosphate pathway is operating in M. sedula, transcriptional patterns of these 2 ORFs

(specifically their similar regulation) are consistent with a shared pathway having a branch point off of another major pathway.

125

Autotrophic growth. The autotrophy transcriptome was characterized by the upregulation of several amino acid biosynthesis gene clusters (Table 4.3), including those for nitrogen-assimilating amino acids (glutamine and glutamate synthases, Msed_1612 and

Msed_1884-Msed_1885), sulfur-assimilating amino acids (cystathionine gamma synthase and methionine synthase, Msed_0673 and Msed_2248/Msed_2249), aliphatic amino acids

(citramalate synthase-like ORF, Msed_0623, ilvCHI, Msed_1927-Msed_1929), and positively charged amino acids (acetylglutamate kinase, L-2-aminoadipate N- acetyltransferase, and N2-acetyl-L-lysine aminotransferase/acetylornithine, Msed_0165,

Msed_0168-Msed_0169, and argininosuccinate lyase, Msed_1988). Riboflavin and thiamine biosynthesis ORFs (Msed_0046-Msed_0048, Msed_0906, and Msed_2221) were also observed to be up-regulated on autotrophy compared to heterotrophy and mixotrophy.

It is not yet known how, or in what form, inorganic carbon is assimilated by M. sedula. CO2 could diffuse through the cell membrane, or CO2 or bicarbonate could enter the cell via transporters. Of the annotated transporters in the M. sedula genome, one was of particular interest. Msed_1539 is annotated as a Rh family ammonium transporter

(IPR001905), which includes AmtB ammonium transporters in bacteria recently suggested to channel ammonia gas. Rh proteins in the green alga Chlamydomonas reinhardtii most likely function as CO2 gas channels (20). Msed_1539 was significantly up-regulated under autotrophic growth compared to either heterotrophic (4.3-fold with p-value 5.0 x 10-13) or mixotrophic (4.3-fold with p-value 2.5 x 10-15) growth conditions and merits further attention for a possible role in CO2 fixation.

126 Carbonic anhydrase catalyze the interconversion of CO2 into bicarbonate.

Msed_0390 (β-class) and Msed_1618 (γ-class) appear to be carbonic anhydrase homologs, although only Msed_0390 was up-regulated under autotrophic conditions compared to heterotrophic conditions (see Table 4.A3). A similar protein in Acidianus ambivalens

(CAC83596, 36% aa identity) was previously characterized as a soluble cytochrome b; however, no activity was detected when the protein was tested for carbonic anhydrase function (8). The β-carbonic anhydrase from the thermophilic archaeon Methanobacterium thermoautotrophicum was activated by some amino acids, although the significance of this finding was not determined (15). Msed_0391, annotated as an amino acid transporter-like protein, lies on the strand opposite the β-carbonic anhydrase, and was up-regulated during autotrophic and mixotrophic growth compared to heterotrophic growth. Its role in CO2 fixation should be considered.

Inorganic carbon fixation. Figure 4.4 lists the enzyme candidates of the recently proposed 3-hydroxypropionate/4-hydroxybutyrate cycle in M. sedula (5), along with the normalized transcription levels for each candidate ORF as a function of autotrophic, heterotrophic, or mixotrophic growth modes (Table 4.A4 lists the same candidates with transcription information presented in differential form, highlighting response of ORFs believed to be statistically significant). As expected, the known inorganic carbon fixation cycle enzymes were up-regulated on autotrophic conditions compared to heterotrophic conditions, including malonyl-CoA reductase, which was the largest change of ORFs in the cycle and one of the largest of the dataset (Msed_0709: 24.6-fold, with p-value 1.9 x 10-18).

Two exceptions included 3-hydroxypropionyl-CoA dehydratase (Msed_2001) and

127 methylmalonyl epimerase (Msed_0639), which were not differentially transcribed, although these ORFs were transcribed at relatively high levels (lsms of 1.4-2.5 and 0.7-1.8) on all conditions compared to the overall average transcription levels for all ORFs (log2 average =

0). Along with up-regulation of the second subunit of the methylmalonyl-CoA mutase

(Msed_2055), the overlapping Msed_2056 also was highest during autotrophic growth.

Msed_2056 is annotated as a lysine-arginine-ornithine transport system ATPase; proteins of this type (ArgK) have previously been reported to act as kinases, phosphorylating bacterial periplasmic transport proteins (ArgT) but are not necessarily involved with the transport system function (7). In the case of a putative 4-hydroxybutyryl-CoA dehydratase

(step 11, Msed_1220 or Msed_1321), only Msed_1321 clearly shows significant upregulation on autotrophic vs. heterotrophic conditions, supporting its proposed functional role as the 4-hydroxybutyryl-CoA dehydratase (12). Several gene candidates were proposed as putative crotonyl-CoA hydratases (step 12); however, none were higher on autotrophy vs. heterotrophy. In the case of a putative 3-hydroxybutyryl-CoA dehydrogenase

(step 13), Msed_1993 responded to autotrophy. On the strand opposite of Msed_1993, a cupin superfamily protein (Msed_1994) was highest under autotrophy. The cupin barrel fold has been associated with oxalate decarboxylase/oxidase activity; however, the sequence is also related to COG3435 for 1, 2 gentisate dioygenase, typically involved with tyrosine degradation through de-cyclizing a ring structure into an acetoacetate compound. Of the candidates proposed for the putative acetoacetyl-CoA β-ketothiolase, Msed_0656 was the only ORF that exhibited up-regulation on autotrophy. Also, note that Msed_2087, annotated as an acyl-CoA dehydrogenase domain-containing protein, was not thought to be involved involved in the carbon-fixation cycle. However, its transcriptional patterns match other

128 enzymes in the cycle (up-regulated 7.6-fold on A vs H and 7.8-fold on A vs. M, with p- values of 1.6 x 10-10 and 2.8 x 10-12, respectively), meriting further investigation of the autotrophy-related function of this enzyme.

Hydrogenases. Two different loci in the M. sedula genome appear to encode NiFe hydrogenases. Msed_0913-Msed_0950 encode 2 hydrogenases (3 putative alpha subunits and 2 beta subunits), multiple accessory proteins (HypAC1C2C3DEF and a maturation protease), and multiple hypothetical proteins for which no GenBank database matches have been identified. Msed_1428-1433 appear to encode a third hydrogenase. However, when ClustalW was used to align these 4 potential alpha subunits with previously characterized hydrogenases to look for the canonical Ni-binding motifs involving 4 Cys (28), only Msed_0923 and Msed_0945 met the criteria as hydrogenases (Figure 4.A1). NiFe

Hydrogenases have been classified into 4 basic categories (37): membrane-bound uptake hydrogenases (Group I), cytoplasmic hydrogenases serving an uptake or regulatory hydrogen-sensing function (Group II), bidirectional hydrogenases binding soluble cofactors

(Group III), and the hydrogen-evolving membrane bound hydrogenases having similarities to the multi-subunit NADH oxidoreductases (group IV). Archaeal hydrogenases have been identified for all categories, except Group II (36). The only thermoacidophilic archaeal hydrogenase yet characterized is the Group I HynSL-IspI from A. ambivalens (21).

Msed_0944-Msed_0945 appear to encode the small and large subunits (HynSL;

33% and 28% aa identity, respectively) of a NiFe hydrogenase, while Msed_0946-

Msed_0947 are similar to the Isp1 cytochrome b membrane anchor (22% aa identity) and isp2 Fe-S oxidoreductase-like protein (24% aa identity) of the thermoacidophilic group I

129 NiFe hydrogenase, previously described in Acidianus ambivalens (21). The only other archaeal sequence gene with similarities to Group I NiFe hydrogenases comes from the recently sequenced genome of Sulfolobus islandicus U.3.28 (contig56_2390). A phylogram of beta subunits (similar to those in (21, 37)) suggests that both of these thermoacidophilic hydrogenase sequences should be classified as Group I (see Figure 4.A2).

ORFs Msed_0923-Msed_0924 are also homologous to the beta and alpha subunits of NiFe hydrogenases. Neither signal peptides nor transmembrane helices are predicted for the beta subunit, suggesting that the hydrogenase encoded by Msed_0923-Msed_0924 is cytoplasmic rather than membrane-bound. Phylogenetic analysis suggests that this hydrogenase should be classified as a Group II hydrogenase (see Figure 4.A2). Sulfolobus islandicus U.3.28 has the only other identified archaeal sequences (contig56_2375/_2376) with similarity to Msed_0923-Msed_0924. Both of the beta subunit sequences group more closely with the cyanobacterial uptake HupS sequences than with the hydrogen-sensing

HupU sequences (Msed_0924 has 52% identity to the beta subunit of Anabaena variabilis,

Ava_4596, vs. 39% identity with HupU of Bradyrhizobium japonicum, bll6944). Furthermore, both genomes lack sequences with significant homology to a HupT-like regulatory protein found with bacterial hydrogen-sensing hydrogenases (although it is possible that a different regulatory protein could be present in archaeal systems). The similarly grouped cytoplasmic

Group II hydrogenase of Aquifex aeolicus (aq_802 has 41% identity to Msed_0924) has been suggested to function as an uptake hydrogenase during CO2 fixation (9).

Only one hydrogenase responded when M. sedula was grown aerobically in the presence of hydrogen (see Figure 4.5 and Table 4.A5). The membrane-bound hydrogenase was significantly up-regulated under autotrophy compared to heterotropy (Msed_0944-

130 Msed_0946: 7.4 to 12.6-fold with p-values 8.1 x 10-11 to 2.8 x 10-13). Multiple Hyp-encoding

ORFs, an FeS oxidoreductase-like protein and several hypothetical proteins in the same region also had similar response patterns. The lack of response from the ORFs encoding the soluble hydrogenase suggested that this enzyme may not be activated by the presence of H2 in an aerobic environment, and therefore, not function as uptake hydrogenase.

However, the normalized transcript levels detected for these ORFs (lsmeans~ -1) were lower than the overall average transcript expression levels (average ~0), which is consistent with the low synthesis levels reported for the group II, H2-sensing regulatory hydrogenase (HupUV) in R. capsulatus (38).

The Group I uptake hydrogenases have an energy-conservation function, contributing to the generation of proton motive force by transferring electrons, via quinones, to cytochromes or heterodisulfide reductases with proton pumping capabilities (14, 36, 37).

Both the M. sedula SoxABCDD’ quinol/terminal oxidase cluster (Msed_0285-Msed_0292) and components of the heterodisulfide-like cluster (Msed1542-Msed1550), previously reported to be up-regulated in the presence of reduced inorganic sulfur compounds (RISCs)

(2), were also found to be up-regulated here on H2 (Table 4.A6). In A. aeolicus, sulfur reductase (Sre-like subunits) was found in a super complex with a Group I hydrogenase and bc1 components during growth on elemental sulfur and hydrogen (10). In M. sedula, the sre-like complex (Msed_0810-Msed_0818) was found to be up-regulated on RISCs (2), but not in the presence of hydrogen (Table 4.A6). The Sre complex studied in A. ambivalens was inactive unless it was also coupled with the Group I hydrogenase (21). A review of the

M. sedula Group I hydrogenase transcriptional patterns in the presence of elemental sulfur

(GEO accession 12044) shows that both the alpha subunit and the cytochrome b

131 membrane anchor (Msed_0945-Msed_0946) were stimulated by elemental sulfur compared to the absence of elemental sulfur, the presence of Fe(II), or the presence of more oxidized forms of sulfur (tetrathionate or sulfate) (Table 4.A7). The cytoplasmic ferredoxin/isp2-like transcript (Msed_0947), potentially involved with electron transfer from the hydrogenase, was observed to be down-regulated in the presence of elemental sulfur compared to the same conditions.

Mixotrophic growth. Very few ORFs uniquely responded to mixotrophy; Table 4.4 includes the 15 ORFs that were up-regulated on mixotrophy compared to autotrophy and heterotrophy, as well as 9 ORFs that were down-regulated on mixotrophy compared to autotrophy and heterotrophy. Of these two dozen ORFs, 5 are annotated as hypothetical proteins, although in the case of Msed_0849, closer inspection show that this ORF most likely encodes the inner membrane/permease component of an ABC transporter. Seven

ORFs appear to have roles in electron transfer. Of the ORFs showing up-regulation on mixotrophy, Msed_0428 is annotated as an NADPH-dependent FMN reductase and has similarity (35% aa identity) to the WrbA flavoprotein, which is involved with quinone reduction in hyperthermophilic euryarchaeon Archaeoglobus fulgidis, and has been proposed to be a 4th category of NAD(P)H:quinone oxidoreductase (NDH-IV) (25).

Msed_0500 (soxN) and Msed_0502 (cbsA) are putative cytochromes b located in a cluster of ORFs possibly involved with dissimilatory electron transfer from inorganic energy sources (2, 17). Msed_0997 has similarity (82% aa identity) to sulredoxin, a soluble Rieske

(2Fe-2S) protein that has been biochemically characterized in S. tokodaii, although its specific physiological role remains unclear (16). Electron transfer-associated ORFs which

132 are down-regulated on mixotrophy include: soxM (Msed_0324), part of the SoxEFGHIM terminal oxidase complex, which has previously been suggested to be involved with electron transfer under heterotrophic growth conditions (17), a FAD-dependent pyridine nucleotide disulfide oxidoreductase (Msed_0743), and the δ subunit of a pyruvate:ferredoxin/flavodoxin oxidoreductase complex (PorD, Msed_1201). Most of the other ORFs that responded to mixotrophy also responded to either autotrophic or heterotrophic growth conditions, supporting the idea that mixotrophy is the simultaneous use or combination of various systems utilized during autotrophic and heterotrophic growth.

The ORFs of the 3-hydroxpropionate/4-hydroxybutyrate cycle were transcribed at lower levels under mixotrophy compared to autotrophy, with the exception of the 3- hydroxypropionyl-CoA dehydratase (Msed_2001), the acroloyl-CoA reductase

(Msed_1426), the methylmalonyl-CoA epimerase (Msed_0639), and the succinate semialdehyde reductase (Msed_1424). Malonyl-/succinyl-CoA reductase (Msed_0709) and the acroloyl reductase (Msed_1426) were transcribed at higher levels during mixotrophic vs heterotrophic growth (Msed_709: 2.4-fold with p-value 2.8 x 10-4, Msed_1426: 1.8-fold with p-value 1.6 x 10-3), suggesting that some inorganic carbon may still be assimilated during mixotrophic growth (note that heterotrophic growth conditions still contain whatever CO2 is in the air used to sparge the culture). However, a strong shift away from inorganic carbon fixation during mixotrophy was observed.

To be growing mixotrophically (according to definition #2), M. sedula should be utilizing organic carbon from tryptone and electrons exclusively from inorganic hydrogen.

The membrane-bound hydrogenase αβ subunits (Msed_0944-Msed_0945) were significantly up-regulated on mixotrophy compared to heterotrophy, although two presumed

133 electron-transferring proteins, membrane anchor (isp1, Msed_0946) and cytoplasmic Fe-S oxidoreductase like protein (isp2, Msed_0947), as well as hypC3E (Msed_0931 and

Msed_0933), several associated hypotheticals (Msed_0932, Msed_0940, Msed_0949), and a putative amino-acid permease (Msed_0935) were down-regulated on mixotrophy compared to autotrophy (Table 4.A5). Taken together, this data indicates that while H2 is being utilized during mixotrophic growth, it is to a lesser extent than during autotrophic growth. This implies that organic carbon may also be serving as an energy source during mixotrophic growth.

Hypothetical proteins. A large number of hypothetical ORFs were also observed to be differentially transcribed under the various growth conditions, indicating there is more to the story than can be gleaned from current information. For example, three transporters were observed to be up-regulated on autotrophic conditions compared to heterotrophy or mixotrophy: major facilitator superfamily transporters (Msed_0254: up 20.7-fold (p-value 1.6 x 10-21) on A vs. H and 23.5-fold (p-value 5.6 x 10-23) on A vs. M, Msed_0426: up 5.0-fold

(p-value 1.4 x 10-10) on A vs. H and 7.3-fold (p-value 7.6 x 1014) on A vs M, Msed_0463: up

4.1-fold (p-value 3.7 x 10-06) on A vs H and 2.9-fold (p-value 1.1 x 10-04) on A vs M, and

Msed_0467: up 10.1-fold (p-value 3.4 x 10-17) on A vs H), amino acid permeases

(Msed_1269, Msed_1312, and Msed_1503), and an ABC transporter (Msed_1286-

Msed_1289). One potential operon, containing a rhodanese-like protein (Msed_1209-

Msed_1211), was up-regulated 2.5 to 7.0–fold (p-values 3.1 x 10-05 to 1.1 x 10-14) under similar conditions.

134 Summary. In this work, various aspects of M. sedula physiology were examined under heterotrophic, autotrophic, and mixotrophic growth conditions. In examining central carbon metabolism, minimal regulation of gluconeogenesis and the TCA cycle were observed, except at points of entry into gluconeogenesis (acetyl-CoA and pyruvate) and at the point of the TCA cycle’s succinyl-CoA intermediate, which is also common to the CO2- fixing 3-hydroxypropionate/4-hydroxybutyrate cycle. Transcriptional patterns of the known/characterized enzymes of this cycle were consistent with inorganic carbon fixation under autotrophic growth and several ORF candidates proposed for uncharacterized enzymes of the cycle were confirmed or ruled out. Observation of CO2-fixation cycle transcripts on mixotrophic growth suggested M. sedula has a strong preference for organic carbon under the conditions tested. The membrane-bound hydrogenase responsible for allowing M. sedula autotrophic growth on a H2-based inorganic energy source was identified, and potential routes for contribution to the generation of ATP (via proton motive force) and reducing equivalents were suggested. Transcripts indicated H2 was utilized as an energy source during mixotrophic growth, although the highest levels occurred under autotrophic growth, potentially implicating organic carbon as an additional energy source during mixotrophic growth. The faster M. sedula growth rates observed under mixotrophic growth conditions are most likely to be the result of optimizing the carbon and energy sources available.

135 ACKNOWLEDGEMENTS

We would like to thank MWW Adams, University of Georgia, for helpful discussions regarding the manuscript. KSA acknowledges support from an NIH T32 Biotechnology

Traineeship. RMK acknowledges support from the NSF Biotechnology Program.

REFERENCES

1. Alber, B., M. Olinger, A. Rieder, D. Kockelkorn, B. Jobst, M. Hugler, and G. Fuchs. 2006. Malonyl-coenzyme A reductase in the modified 3-hydroxypropionate cycle for autotrophic carbon fixation in archaeal Metallosphaera and Sulfolobus spp. J. Bacteriol. 188:8551-9.

2. Auernik, K. S., and R. M. Kelly. 2008. Identification of components of electron transport chains in the extremely thermoacidophilic crenarchaeon Metallosphaera sedula through iron and sulfur compound oxidation transcriptomes. Appl. Environ. Microbiol. 74:7723-32.

3. Auernik, K. S., Y. Maezato, P. H. Blum, and R. M. Kelly. 2008. The genome sequence of the metal-mobilizing, extremely thermoacidophilic archaeon Metallosphaera sedula provides insights into bioleaching-associated metabolism. Appl. Environ. Microbiol. 74:682-92.

4. Bagos, P. G., K. D. Tsirigos, S. K. Plessas, T. D. Liakopoulos, and S. J. Hamodrakas. 2009. Prediction of signal peptides in archaea. Protein Eng. Des. Sel. 22:27-35.

5. Berg, I. A., D. Kockelkorn, W. Buckel, and G. Fuchs. 2007. A 3- hydroxypropionate/4-hydroxybutyrate autotrophic carbon dioxide assimilation pathway in Archaea. Science 318:1782-6.

6. Boyle, N. R., and J. A. Morgan. 2009. Flux balance analysis of primary metabolism in Chlamydomonas reinhardtii. BMC Syst. Biol. 3:4.

136 7. Celis, R. T., P. F. Leadlay, I. Roy, and A. Hansen. 1998. Phosphorylation of the periplasmic binding protein in two transport systems for arginine incorporation in Escherichia coli K-12 is unrelated to the function of the transport system. J. Bacteriol. 180:4828-33.

8. Gomes, C. M., A. Kletzin, and M. Teixeira. 2002. An archaeal b-type cytochrome containing a nonfunctional carbonic anhydrase-like domain. J. Biol. Inorg. Chem. 7:483-9.

9. Guiral, M., C. Aubert, and M. T. Giudici-Orticoni. 2005. Hydrogen metabolism in the hyperthermophilic bacterium Aquifex aeolicus. Biochem. Soc. Trans. 33:22-4.

10. Guiral, M., P. Tron, C. Aubert, A. Gloter, C. Iobbi-Nivol, and M. T. Giudici- Orticoni. 2005. A membrane-bound multienzyme, hydrogen-oxidizing, and sulfur- reducing complex from the hyperthermophilic bacterium Aquifex aeolicus. J. Biol. Chem. 280:42004-15.

11. Han, C. J., and R. M. Kelly. 1998. Biooxidation capacity of the extremely thermoacidophilic archaeon Metallosphaera sedula under bioenergetic challenge. Biotechnol. Bioeng. 58:617-24.

12. Huber, H., M. Gallenberger, U. Jahn, E. Eylert, I. A. Berg, D. Kockelkorn, W. Eisenreich, and G. Fuchs. 2008. A dicarboxylate/4-hydroxybutyrate autotrophic carbon assimilation cycle in the hyperthermophilic Archaeum Ignicoccus hospitalis. Proc. Natl. Acad. Sci. U. S. A. 105:7851-6.

13. Hugler, M., R. S. Krieger, M. Jahn, and G. Fuchs. 2003. Characterization of acetyl-CoA/propionyl-CoA carboxylase in Metallosphaera sedula. Carboxylating enzyme in the 3-hydroxypropionate cycle for autotrophic carbon fixation. Eur. J. Biochem. 270:736-44.

14. Ide, T., S. Baumer, and U. Deppenmeier. 1999. Energy conservation by the H2:heterodisulfide oxidoreductase from Methanosarcina mazei Go1: identification of two proton-translocating segments. J. Bacteriol. 181:4076-80.

15. Innocenti, A., S. A. Zimmerman, A. Scozzafava, J. G. Ferry, and C. T. Supuran. 2008. Carbonic anhydrase activators: activation of the archaeal beta-class (Cab) and gamma-class (Cam) carbonic anhydrases with amino acids and amines. Bioorg. Med. Chem. Lett. 18:6194-8.

137 16. Iwasaki, T., T. Imai, A. Urushiyama, and T. Oshima. 1996. Redox-linked ionization of sulredoxin, an archaeal Rieske-type [2Fe-2S] protein from Sulfolobus sp. strain 7. J. Biol. Chem. 271:27659-63.

17. Kappler, U., L. I. Sly, and A. G. McEwan. 2005. Respiratory gene clusters of Metallosphaera sedula - differential expression and transcriptional organization. Microbiology 151:35-43.

18. Kardinahl, S., C. L. Schmidt, T. Hansen, S. Anemuller, A. Petersen, and G. Schafer. 1999. The strict molybdate-dependence of glucose-degradation by the thermoacidophile Sulfolobus acidocaldarius reveals the first crenarchaeotic molybdenum containing enzyme--an aldehyde oxidoreductase. Eur. J. Biochem. 260:540-8.

19. Koide, A., and J. A. Hoch. 1994. Identification of a second oligopeptide transport system in Bacillus subtilis and determination of its role in sporulation. Mol. Microbiol. 13:417-26.

20. Kustu, S., and W. Inwood. 2006. Biological gas channels for NH3 and CO2: evidence that Rh (Rhesus) proteins are CO2 channels. Transfus. Clin. Biol. 13:103- 10.

21. Laska, S., F. Lottspeich, and A. Kletzin. 2003. Membrane-bound hydrogenase and sulfur reductase of the hyperthermophilic and acidophilic archaeon Acidianus ambivalens. Microbiology 149:2357-71.

22. Orita, I., T. Sato, H. Yurimoto, N. Kato, H. Atomi, T. Imanaka, and Y. Sakai. 2006. The ribulose monophosphate pathway substitutes for the missing pentose phosphate pathway in the archaeon Thermococcus kodakaraensis. J. Bacteriol. 188:4698-704.

23. Padden, A. N., D. P. Kelly, and A. P. Wood. 1998. Chemolithoautotrophy and mixotrophy in the thiophene-2-carboxylic acid-utilizing Xanthobacter tagetidis. Arch. Microbiol. 169:249-56.

24. Palmieri, G., A. Casbarra, I. Fiume, G. Catara, A. Capasso, G. Marino, S. Onesti, and M. Rossi. 2006. Identification of the first archaeal oligopeptide-binding protein from the hyperthermophile Aeropyrum pernix. Extremophiles 10:393-402.

138 25. Patridge, E. V., and J. G. Ferry. 2006. WrbA from Escherichia coli and Archaeoglobus fulgidus is an NAD(P)H:quinone oxidoreductase. J. Bacteriol. 188:3498-506.

26. Peeples, T. L., and R. M. Kelly. 1995. Bioenergetic Response of the Extreme Thermoacidophile Metallosphaera sedula to Thermal and Nutritional Stresses. Appl. Environ. Microbiol. 61:2314-2321.

27. Perego, M., C. F. Higgins, S. R. Pearce, M. P. Gallagher, and J. A. Hoch. 1991. The oligopeptide transport system of Bacillus subtilis plays a role in the initiation of sporulation. Mol. Microbiol. 5:173-85.

28. Przybyla, A. E., J. Robbins, N. Menon, and H. D. Peck, Jr. 1992. Structure- function relationships among the nickel-containing hydrogenases. FEMS Microbiol. Rev. 8:109-35.

29. Rouillard, J. M., M. Zuker, and E. Gulari. 2003. OligoArray 2.0: design of oligonucleotide probes for DNA microarrays using a thermodynamic approach. Nucleic Acids Res. 31:3057-62.

30. Selosse, M. A., and M. Roy. 2009. Green plants that feed on fungi: facts and questions about mixotrophy. Trends Plant Sci. 14:64-70.

31. Shively, J. M., G. van Keulen, and W. G. Meijer. 1998. Something from almost nothing: carbon dioxide fixation in chemoautotrophs. Annu. Rev. Microbiol. 52:191- 230.

32. Snijders, A. P., J. Walther, S. Peter, I. Kinnman, M. G. de Vos, H. J. van de Werken, S. J. Brouns, J. van der Oost, and P. C. Wright. 2006. Reconstruction of central carbon metabolism in Sulfolobus solfataricus using a two-dimensional gel electrophoresis map, stable isotope labelling and DNA microarray analysis. Proteomics 6:1518-29.

33. Starkenburg, S. R., D. J. Arp, and P. J. Bottomley. 2008. D-Lactate metabolism and the obligate requirement for CO2 during growth on nitrite by the facultative lithoautotroph Nitrobacter hamburgensis. Microbiology 154:2473-81.

139 34. Tittel, J., V. Bissinger, U. Gaedke, and N. Kamjunke. 2005. Inorganic carbon limitation and mixotrophic growth in Chlamydomonas from an acidic mining lake. Protist 156:63-75.

35. Tittel, J., V. Bissinger, B. Zippel, U. Gaedke, E. Bell, A. Lorke, and N. Kamjunke. 2003. Mixotrophs combine resource use to outcompete specialists: implications for aquatic food webs. Proc. Natl. Acad. Sci. U. S. A. 100:12776-81.

36. Vignais, P. M. 2008. Hydrogenases and H(+)-reduction in primary energy conservation. Results Probl. Cell Differ. 45:223-52.

37. Vignais, P. M., B. Billoud, and J. Meyer. 2001. Classification and phylogeny of hydrogenases. FEMS Microbiol. Rev. 25:455-501.

38. Vignais, P. M., S. Elsen, and A. Colbeau. 2005. Transcriptional regulation of the uptake [NiFe]hydrogenase genes in Rhodobacter capsulatus. Biochem. Soc. Trans. 33:28-32.

140

Table 4.1: Occurrence of select genes in extremely thermoacidophilic genomes implicated in processing varied carbon and energy sources. Components Ss St Sa Pt Ms Cm SiL SiM SiU SiYGN Ab Aa Sm Terminal oxidases SoxABCDD’L X X X - X - X X X X - ? ? SoxEFGHIM X X X X X X X X X X - ? ? DoxBCE X X X X X X X X X X - X ? Iron oxidation FoxABCD - X - - X ------? X Rusticyanin1 - - - - X X X X X X - ? ? Rusticyanin2 - - - - X - - - X X - ? ? Sulfur Oxidation Sor - X - X ------X X TetH - X - X X X X X X X - ? ? DoxDA/TQO X X - X X - X X X X - X X CO2 Fixation 3-hydr/4-butyr X X X - X - X X X X - ? ? H2 utilization Hydrogenase - - - - X - ? - X - X X ? (NiFe) Ss (Sulfolobus solfataricus), St (Sulfolobus tokodaii), Sa (Sulfolobus acidocaldarius), Pt (Picrophilus torridus), Ms (Metallosphaera sedula), Cm (Caldivirga maquilingensis), Si (Sulfolobus islandicus, strains L,M,U,YG&YN), Ab (Aciduliprofundum boonei), Aa (Acidianus ambivalens), Sm (Sulfolobus metallicus)

141

Table 4.2: ORFs up-regulated on heterotrophy in M. sedula genome. Significant differential transcription is considered to have occurred where log2 fold change has a magnitude greater than 1 (equivalent to a 2-fold change) with significance at or above the Bonferroni (≥5.4 for this data set). Average of log2 fold changes and significance values are reported for all 4 combinations of biological repeats (i.e. average of log2 fold changes for autotrophy vs heterotrophy averages values for individual comparisons A1-H1, A1-H2, A2-H1, and A2-H2) GenBank Annotation (02Mar2009) Average Average Average msed manual annotation notes Log2 fold change Log2 fold change Log2 fold change ORF (A)-(H) (H)-(M) (A)-(M) # [average Siga] [average Siga] [average Siga]

0268 Hypothetical protein (141aa) -1.0 [1.4] 2.3 [6.5] 1.4 [2.9]

0270 acetyl-CoA acetyltransferase (391aa) -0.9 [1.7] 1.6 [5.3] 0.7 [1.6] non-specific lipid/sterol carrier protein

Hypothetical protein (144aa) -1.7 [7.0] 1.3 [5.7] -0.4 [1.8] 0319 SoxI

Rieske (2Fe-2S) domain-containing -1.0 [1.8] 1.4 [3.1] 0.3 [0.6] 0322 protein (272aa)

SoxF

0333 FAD dependent oxidoreductase (401aa) -2.1 [6.9] 1.4 [4.4] -0.7 [1.6] Flavoprotein dehydrogenase-like protein

0349 Hypothetical protein (240aa) -1.0 [3.4] -1.9 [7.3] 0.8 [1.8]

0850 Hypothetical protein (340aa) -2.3 [9.2] 0.8 [2.0] -1.6 [6.2]

0855 methyltransferase type 11 (181aa) -2.1 [7.8] 1.5 [5.3] -0.6 [2.5] NAD(P)-binding 0858 Hypothetical protein (252aa) -0.8 [8.4] 1.0 [4.9] -0.7 [2.8]

142 (Table 4.2 continued)

0900 Hypothetical protein (127aa) -1.5 [7.5] 1.2 [6.9] -0.2 [0.9]

0993 Major facilitator transporter (497aa) -0.8 [3.1] 1.4 [5.7] 0.6 [3.8] Similarities to organic substrate transporters

1037 acetolactate synthase, large subunit (551aa) -2.6 [6.0] 1.4 [2.4] -1.3 [3.0]

1095 Major facilitator transporter (477aa) -1.9 [7.7] 0.8 [2.6] -1.1 [4.2]

1177 FAD dependent oxidoreductase (435aa) -1.6 [8.1] 1.0 [5.0] -0.6 [2.2] 41% identity to glycerol-3-phosphate dhase in E. coli 1178 glycerophosphodiester phosphodiesterase -1.4 [8.1] 0.2 [0.7] -1.2 [7.5] (245aa) 1180 potentially converts phospholipids to G-3-P -2.3 [7.6] 0.9 [2.4] -1.4 [4.4] ABC transporter related (312aa) similar to ATPase for bacterial G-3-P import system TM helix repeat-containing protein (254aa) 1227 -2.4 [7.4] 1.3 [3.3] -1.1 [3.0] Membrane protein with 6 predicted TMs 1254 Hypothetical protein (135aa) -1.6 [6.0] 1.2 [4.5] -0.4 [2.3] succinyl-CoA synthetase (ADP-forming) α subunit 1581 -1.5 [3.9] 1.3 [3.8] -0.2 [0.8] (263aa)

succinyl-CoA synthetase (ADP-forming)β subunit 1582 -2.3 [5.5] 1.7 [4.4] -0.6 [0.9] (337aa) 1733 transcription initiation factor IIB (294aa) -2.0 [6.3] 1.2 [3.4] -0.8 [2.2] major facilitator transporter (489aa) 1772 -1.1 [5.0] 1.0 [5.2] -0.1 [0.6]

3,4-dihydroxyphenylacetate 2,3-dioxygenase 1775 -2.2 [9.3] 1.8 [8.1] -0.4 [1.4] (314aa) 1822 hypothetical protein (291aa) -1.6 [5.7] 1.2 [4.6] -0.4 [0.7] hypothetical protein (100aa) 1833 -1.9 [6.2] 1.1 [3.1] -0.7 [3.3] Sulfolobus transposase-like domain hypothetical protein (204aa) 1854 -2.0 [7.6] 0.9 [2.5] -1.2 [3.8] similar to bacterial glycosyl hydrolase (family 1) Glu/Leu/Phe/Val dehydrogenase, C terminal (421aa) 2074 -2.1 [5.7] 0.4 [0.6] -1.7 [5.0] similar to Glu dhase in S. solfataricus, Maras et al. 1992 hypothetical protein (249aa) 2282 -1.5 [5.6] 1.1 [3.8] -0.4 [2.2] similar to phage-integrase from SSV1 a Significance = -log10(p-value). For reference, a significance value of 5.4 correlates to a p-value of 4 x 10-6.

143

Table 4.3: Putative biosynthesis ORFs upregulated on autotrophy in M. sedula genome. Significant differential transcription is considered to have occurred where log2 fold change has a magnitude greater than 1 (equivalent to a 2-fold change) with significance at or above the Bonferroni (≥5.4 for this data set). Average of log2 fold changes and significance values are reported for all 4 combinations of biological repeats (i.e. average of log2 fold changes for autotrophy vs heterotrophy averages values for individual comparisons A1-H1, A1-H2, A2-H1, and A2-H2 GenBank Annotation (01Mar2009) Average manual annotation notes Average Average Log2 fold Log2 fold Log2 fold change Msed change change (H)-(M) ORF (A)-(H) (A)-(M) [average # [average Siga] [average Siga] Siga] Nitrogen assimilating amino acids 1612 L-glutamine synthetase (470aa) 2.3 [7.7] 2.1 [7.4] -0.3 [0.6] glnA; similar to S. solfataricus, Sanangelantoni et al. 1990 1884 glutamate synthase (NADPH) GltB2 subunit (712aa) 2.3 [6.0] 2.3 [6.8] 0.0 [1.0]

1885 glutamate synthase (NADPH) GltB1 subunit (646aa) 0.9 [3.7] 1.8 [9.4] 0.9 [3.9] Sulfur assimilating amino acids 0960 uroporphyrin-III /tetrapyrrole methyltransferase 1.9 [5.5] -0.2 [0.7] -2.0 [7.1] (227aa) cysG-like; Auernik et al. 2008 0961 sulfite reductase (NADPH) beta subunit (599aa) 4.6 [18.4] 1.2 [3.8] -3.4 [14.6] cysI-like; Auernik et al. 2008 0962 phosphoadenosine phosphosulfate reductase 3.4 [13.9] 0.8 [2.2] -2.5 [11.2] (238aa) 0963 cysH-like; Auernik et al. 2008 3.2 [10.1] 1.4 [3.4] -1.8 [5.4] sat sulfate adenylyltransferase (369aa) cysN-like; Auernik et al. 2008

0672 3.3 [11.7] 3.4 [14.2] 0.1 [0.3] homoserine kinase (311aa) 0673 also used in threonine biosynthesis 2.7 [7.6] 2.4 [7.4] -0.3 [0.4] hypothetical protein (374aa) cystathionine gamma synthase

2248 0.9 [1.1] 2.3 [5.3] 1.4 [2.6] methionine synthase (331aa) 2249 0.9 [1.7] 2.2 [7.3] 1.2 [3.3] 5-methyltetrahydropteroyltriglutamate-- 2250 homocysteine methyltransferase (320aa) 1.3 [3.0] 2.1 [7.0] 0.8 [1.7] DNA polymerase sliding clamp subunit A (245aa)

144 (Table 4.3 continued)

Aliphatic amino acids 0623 trans-homoaconitate synthase (377aa) 1.9 [5.1] 2.1 [7.1] 0.2 [0.2] LeuA-/CimA-like 1765 2-isopropylmalate synthase (460aa) 2.9 [11.2] 0.7 [1.5] -2.2 [9.1] LeuA-CimA-like

0828 aminotransferase, class V (383aa) 1.2 [2.5] 0.0 [0.4] -1.2 [3.0]

0829 3-isopropylmalate dehydratase small subunit 1.2 [4.3] 0.0 [0.1] -1.2 [5.4] (161aa) 0830 LeuD-like; similar to G. sulfurreducens, Risso et al. 1.6 [3.1] 0.7 [1.1] -0.9 [1.6] 2008 3-isopropylmalate dehydratase large subunit (415aa) 1927 LeuC-like; similar to G. sulfurreducens, Risso et al. 1.2 [2.7] 1.1 [2.8] -0.1 [0.4] 2008 1928 2.0 [5.5] 2.1 [7.1] 0.1 [0.2]

1929 ketol-acid reductoisomerase (335aa) 1.9 [3.6] 1.8 [3.9] -0.1 [0.1] IlvC-like; similar to G. sulfurreducens, Risso et al. 2008 hypothetical protein (128aa) IlvH-like; similar to G. sulfurreducens, Risso et al. 2008 acetolactate synthase catalytic subunit (572aa) IlvI-like; similar to G. sulfurreducens, Risso et al. 2008 Aromatic amino acids 1688 phosphoribosylanthranilate isomerase (194aa) 0.7 [1.5] 1.2 [4.2] 0.5 [1.2] TrpF-like 1689 anthranilate phosphoribosyltransferase (343aa) 0.8 [2.0] 1.1 [3.5] 0.3 [0.8] TrpD-like Hydrophilic (+) R group amino acids 0165 acetylglutamate kinase (262aa) 2.0 [6.5] 2.1 [8.6] 0.1 [0.3] involved in Lys and Arg biosynthesis 0168 L-2-aminoadipate N-acetyltransferase (283aa) 1.9 [4.2] 2.2 [6.2] 0.3 [0.3] LysX-like 0169 N2-acetyl-L-lysine aminotransferase / 1.9 [4.8] 2.1 [6.4] 0.2 [0.3] acetylornithine (386aa); ArgD-like, involved in Lys and Arg 1988 biosynthesis 1.6 [5.1] 0.8 [2.3] -0.8 [2.2]

argininosuccinate lyase (448aa) ArgH-like Riboflavin biosynthesis 0046 3,4-dihydroxy-2-butanone 4-phosphate synthase 2.1 [7.7] 3.1 [14.7] 0.9 [2.8] (220aa) 0047 RibB-like 1.8 [5.6] 2.1 [8.3] 0.3 [0.7] riboflavin synthase (156aa) 0048 RibC-like 1.8 [4.5] 2.6 [9.3] 0.8 [1.6] riboflavin synthase subunit beta (151aa) RibH Thiamine biosynthesis 2221 ribulose-1,5-biphosphate synthetase (271aa) 2.6 [11.3] 3.7 [20.1] 1.1 [4.2] putative thiazole biosynthetic enzyme 0906 thiamine biosynthesis protein ThiC (435aa) 2.8 [13.7] 3.0 [17.5] 0.3 [1.2] a Significance = -log10(p-value). For reference, a significance value of 5.4 correlates to a p-value of 4 x 10-6.

145 Table 4.4: ORFs up-regulated or down-regulated on mixotrophy in M. sedula genome. Significant differential transcription is considered to have occurred where log2 fold change has a magnitude greater than 1 (equivalent to a 2-fold change) with significance at or above the Bonferroni (≥5.4 for this data set). Average of log2 fold changes and significance values are reported for all 4 combinations of biological repeats (i.e. average of log2 fold changes for autotrophy vs heterotrophy averages values for individual comparisons A1-H1, A1-H2, A2-H1, and A2-H2). GenBank Annotation (28Feb2009) Average Average Average manual annotation notes Log2 fold Log2 fold Log2 fold msed change change change ORF (A)-(M) (H)-(M) (A)-(H) # [average Siga] [average Siga] [average Siga]

0056 DKCLD domain containing protein (84aa) -1.2 [3.6] -1.2 [3.5] 0.0 [0.5] pseudouridine synthase (α subunit) 0057 PUA domain containing protein (233aa) -1.9 [7.9] -1.3 [4.8] -0.5 [1.0] pseudouridine synthase (β subunit) 0058 methyltransferase small (186aa) -1.4 [6.3] -1.4 [6.1] 0.0 [0.2] rRNA methyltransferase

0428 NADPH-dependent FMN reductase (197aa) -2.8 [13.3] -2.0 [8.4] -0.8 [2.0] wrbA-like

cytochrome b/b6 domain-containing protein 0500 -1.5 [5.1] -2.7 [10.3] 1.2 [2.5] (548aa)

SoxN cytochrome b -2.3 [7.5] -3.0 [10.8] 0.8 [1.1] 0502 pseudogene Cbs A-like cytochrome b Hypothetical protein (291aa) 0517 -1.0 [3.4] -1.9 [7.3] 0.8 [1.8] NTP-binding NTD

0849 hypothetical protein (220aa) -1.0 [3.9] -1.3 [5.7] 0.3 [0.6] ABC transporter, permease subunit (cobalt?)

Rieske [2Fe-2S] protein (109aa) 0997 -2.7 [14.2] -1.3 [5.0] -1.4 [4.8] sulredoxin (82% ID to S. tokodaii, Iwasaki et al. 1996)

1205 hypothetical protein (95aa) -1.3 [5.8] -1.1 [4.9] -0.2 [0.3] (unique 28-Feb-2009)

1364 GMP synthase, α subunit (189aa) -1.8 [5.8] -1.3 [3.4] -0.5 [0.8]

1365 dual specificity protein phosphatase (147aa) -1.3 [2.6] -0.9 [1.7] -0.3 [0.3] possibly involved with signal transduction?

1366 Endonuclease V (200aa) -0.9 [2.8] -0.8 [2.3] -0.1 [0.5]

2054 hypothetical (95aa) -1.0 [4.7] -1.3 [6.8] 0.3 [1.0] CopG/Arc/MetJ family transcriptional 2232 -1.6 [6.0] -1.4 [4.6] -0.3 [0.9] regulator (48aa)

146

(Table 4.4 continued)

GenBank Annotation (28Feb2009) Average Average manual annotation notes Average Log2 fold Log2 fold Log2 fold change change msed change (H)-(M) (A)-(H) ORF (A)-(M) [average [average # [average Siga] Siga] Siga]

0077 exosome complex exonuclease Rrp41 (245aa) 2.5 [11.1] 1.5 [5.4] 1.0 [2.3] (2p)

0227 orotidine 5'-phosphate decarboxylase (227aa) 2.0 [8.2] 1.7 [6.4] 0.3 [0.7] 3-hexulose 6-phosphate synthase (HPS)

0324 cytochrome c oxidase (801aa) 2.1 [6.4] 2.6 [8.7] -0.5 [0.7] SoxM cytochrome oxidase

0328 hypothetical protein (167aa) 2.1 [9.8] 1.6 [6.9] 0.5 [1.0]

FAD-dependent pyridine nucleotide-disulphide 0743 oxidoreductase (335aa) 3.1 [12.1] 1.3 [3.3] 1.8 [4.7] thioredoxin reductase? pyruvate ferredoxin/flavodoxin oxidoreductase, delta 1201 subunit (362aa) 1.7 [7.2] 1.8 [8.0] -0.1 [0.3] PorD-like at CTD only metallophosphoesterase (278aa) 1491 2.5 [10.9] 1.3 [4.2] 1.2 [3.3] possibly involved with signal transduction? SpoVT/AbrB domain-containing protein (326aa) 1659 2.2 [9.4] 1.9 [7.5] 0.3 [0.5] regulatory protein? ribulose-1,5-biphosphate synthetase (271aa) 2221 thiazole biosynthesis protein 3.7 [20.1] 1.1 [4.2] 2.6 [11.3] a Significance = -log10(p-value). For reference, a significance value of 5.4 correlates to a p-value of 4 x 10-6.

147

Figure 4.1: Growth curves for the 3rd passage of heterotrophic (●), autotrophic (), and mixotrophic () cultures of M. sedula. Biological repeats are represented by open shapes and dashed lines. Error bars represent σ.

148

Figure 4.2: Transcriptional levels of M. sedula ORFs implicated in TCA cycle operation (based on model in S. solfataricus (31)) during autotrophic (A1 and A2), heterotrophic (H1 and H2), and mixotrophic (M1 and M2) growth. Heat plots were constructed using AFM 4.0. Red indicates high transcription and green indicates low transcription, and corresponding numbers represent least square means of normalized log2 transformed transcription levels relative to the overall average transcription level of 0. PEP = phosphoenolpyruvate.

149

Figure 4.3: Transcriptional levels of M. sedula ORFs implicated in gluconeogenesis (based on model in S. solfataricus (31) and T. kodakarensis (22)) during autotrophic (A1 and A2), heterotrophic (H1 and H2), and mixotrophic (M1 and M2) growth. Heat plots were constructed using AFM 4.0. Red indicates high transcription and green indicates low transcription, and corresponding numbers represent least square means of normalized log2 transformed transcription levels relative to the overall average transcription level of 0. G-3-P = glyceraldehyde-3-phosphate; 1,3-bisPG =1,3 bisphopoglycerate; 3-PG = 3- phosphoglycerate; 2-PG = 2-phosphoglycerate; PEP = phosphoenolpyruvate.

150

Figure 4.4: Transcriptional levels of M. sedula ORFs implicated in the 3- hydroxpropionate/4-hydroxbutyrate cycle (based M. sedula model proposed by (5)) during heterotrophic (H1 and H2), autotrophic (A1 and A2), and mixotrophic (M1 and M2) growth. Heat plots were constructed using AFM 4.0. Red indicates high transcription and green indicates low transcription, and corresponding numbers represent least square means of normalized log2 transformed transcription levels relative to the overall average transcription level of 0. Enzymes: 1 = acetyl-CoA/propionyl-CoA carboxylase, 2 = malonyl-CoA/succinyl- CoA reductase, 3 = malonate semialdehyde reductase, 4 = 3-hydroxypropionyl-CoA synthetase, 5 = 3-hydroxypropionyl-CoA dehydratase, 6 = acryloyl-CoA reductase, 7 = methylmalonyl-CoA epimerase, 8 = methylmalonyl-CoA mutase, 9 = succinate semialdehyde reductase, 10 = 4-hydroxybutyryl-CoA synthetase, 11 = 4-hydroxybutyryl- CoA dehydratase, 12 = crotonyl-CoA hydratase, 13 = (S)-3-hydroxybutyryl-CoA dehydrogenase, 14 = acetoacetyl-CoA β-ketothiolase.

151

Figure 4.5: Transcriptional levels of M. sedula hydrogenase-associated ORFs during heterotrophic (H1 and H2), autotrophic (A1 and A2), and mixotrophic (M1 and M2) growth. Heat plots were constructed using AFM 4.0. Red indicates high transcription and green indicates low transcription, and corresponding numbers represent least square means of normalized log2 transformed transcription levels relative to the overall average transcription level of 0.

152 Chapter 4 Appendix

Physiological versatility of the extremely thermoacidophilic archaeon Metallosphaera sedula supported by heterotrophy, autotrophy and mixotrophy transcriptomes

153

Table 4.A1: Annotated oligo-/di-peptide transporters in M. sedula.

ORF Size TMM/ GenBank Annotation Hits in Swiss Prot (Pubmed ref#) # (aa) SP 0440 536 0 peptidase S9 prolyl oligopeptidase NT WD40-like beta propeller repeats CT A. pernix peptidase (12037315) 0441 760 1 + SP ABC-type dipeptide transporter, Bacterial SBP_ family 5 periplasmic component 0442 382 1 Hypothetical protein 27%ID to unchar ABC transporter ATP-binding protein in E. coli 0443 318 6 binding-protein transport systems inner Permease / TM component membrane component 0444 470 5 + SP binding-protein transport systems inner 37% ID to B. subtilis AppC oligo transporter permease/TM ( membrane component 7997159) 0445 328 0 oligopeptide/dipeptide ABC transporter, 44% ID to B. subtilis OppD oligo transporter premease ATPase subunit (1901616) 0446 313 0 oligopeptide/dipeptide ABC transporter, 40%ID to B. subtilis OppF/AppF ATPase subunit 0447 169 3 Hypothetical protein No significant hits 1046 675 0 + SP extracellular solute-binding protein Bacterial SBP_ family 5; 24% ID to bacterial glutathione- binding proteins 1047 355 6 + SP binding-protein transport systems inner 33%ID to B. subtilis AppB membrane component 291 5 + SP binding-protein transport systems inner 40% ID to E. coli DdpC 1048 membrane component 1049 330 0 oligopeptide/dipeptide ABC transporter, 44%ID to E. coli OppD ATPase subunit 1050 310 0 oligopeptide/dipeptide ABC transporter, 44%ID to B. subtilis AppF ATPase subunit 1051 431 0 ATPase PFAM0637 archaeal ATPase 1052 310 0 peptidase M19, renal dipeptidase NTD: similarity to section of Alanine racemase CTD: 35%ID to S. pombe dipeptidase 1379 267 0 Hypothetical protein Similar to ATPase subunits ~20-25%ID (DdpD) 1380 284 0 ABC transporter related 28% ID to AppD B. subtilis ; ATPase 1381 249 6 ABC transporter di-/oligopeptide/Ni 28% ID to middle region of DppC (1766370) permease protein 400 7 + SP binding-protein transport systems inner NTD: similarity to Aspartate binding region of asp carbamoyl 1382 membrane component transferase CTD: similar to oligo-/dipeptidase

2203 872 0 + SP extracellular solute-binding protein CTD: 21%ID to B. subtilis OppA 2204 347 6 + SP binding-protein transport systems inner 35% ID to B. subtilis OppB membrane component 2205 298 6 binding-protein transport systems inner 31% ID to B. subtilis OppC membrane component 2206 453 0 oligopeptide/dipeptide ABC transporter, NTD is 51% ID ATPase subunit CTD is 29% ID to B. subtilis AppD 2207 416 0 oligopeptide/dipeptide ABC transporter, 45% ID to B. subtilis AppF ATPase subunit

154

Table 4.A2: Putative TCA cycle ORFs (including pyruvate: ferredoxin oxidoreductase – like sequences) demonstrating differential transcription. Significant differential transcription is considered to have occurred where log2 fold change has a magnitude greater than 1 (equivalent to a 2-fold change) with significance at or above the Bonferroni (≥5.4 for this data set). Average of log2 fold changes and significance values are reported for all 4 combinations of biological repeats (i.e. average of log2 fold changes for autotrophy vs heterotrophy averages values for individual comparisons A1-H1, A1-H2, A2-H1, and A2-H2) Average Average Average Log2 fold change Log2 fold change Log2 fold change (A)-(H) (A)-(M) (H)-(M) Enzyme Candidates [average Siga] [average Siga] [average Siga] 2-oxoglutarate: ferredoxin oxidoreductase msed_0524 (β) -1.7 -1.0 0.7 [4.2] [2.2] [1.4] msed_0525 (γα) -1.7 -0.2 1.5 [2.7] [0.2] [2.6] succinyl-CoA synthetase msed_1581 -1.5 -0.2 1.3 [3.9] [0.8] [3.8] msed_1582 -2.3 -0.6 1.7 [5.5] [0.9] [4.4]

putative pyruvate: ferredoxin oxidoreductase msed_0507 (γ) 1.1 -0.2 -1.3 [2.6] [1.7] [4.2] msed_0508 (δ) 1.8 -0.3 -2.0 [4.1] [1.9] [6.3] msed_0509 (α) 1.4 -0.1 -1.5 [4.0] [1.4] [5.4] msed_0510 (β) 2.2 -0.4 -2.6 [6.8] [3.0] [10.0] putative pyruvate: ferredoxin oxidoreductase msed_1199 (β) 0.0 0.8 0.9 [0.3] [2.9] [3.2] msed_1200 (α) -0.2 0.5 0.7 [0.7] [1.6] [2.2] msed_1201 (δ) -0.1 1.7 1.8 [0.3] [7.2] [8.0] putative pyruvate: ferredoxin oxidoreductase msed_1596 (γ) -2.4 -2.2 0.2 [6.1] [6.4] [0.5] msed_1597 (δα) -1.5 -1.1 0.4 [4.1] [3.0] [0.7] msed_1598 (γ+HTH motif) -1.0 -0.7 0.3 [2.6] [2.0] [0.6] a Significance = -log10(p-value). For reference, a significance value of 5.4 correlates to a p-value of 4 x 10-6.

155

Table 4.A3: Carbonic anhydrases annotated in the M. sedula genome. Significant differential transcription is considered to have occurred where log2 fold change has a magnitude greater than 1 (equivalent to a 2-fold change) with significance at or above the Bonferroni (≥5.4 for this data set). Average of log2 fold changes and significance values are reported for all 4 combinations of biological repeats (i.e. average of log2 fold changes for autotrophy vs heterotrophy averages values for individual comparisons A1-H1, A1-H2, A2-H1, and A2-H2) Average Average Average Log2 fold change Log2 fold change Log2 fold change (A)-(H) (A)-(M) (H)-(M) Enzyme Candidates [average Siga] [average Siga] [average Siga]

β class carbonic anhydrase 1.6 1.2 -0.3 msed_0390 [2.4] [2.1] [0.3]

γ class carbonic anhydrase 0.3 -0.2 -0.5 msed_1618 [0.5] [0.6] [0.7]

a Significance = -log10(p-value). For reference, a significance value of 5.4 correlates to a p-value of 4 x 10-6.

156

Table 4.A4: Potential ORFs involved with CO2 fixation via the 3-hydroxypropionate/4-hydroxybutyrate cycle. Significant differential transcription is considered to have occurred where log2 fold change has a magnitude greater than 1 (equivalent to a 2-fold change) with significance at or above the Bonferroni (≥5.4 for this data set). Average of log2 fold changes and significance values are reported for all 4 combinations of biological repeats (i.e. average of log2 fold changes for autotrophy vs heterotrophy averages values for individual comparisons A1-H1, A1-H2, A2-H1, and A2-H2) Average Average Average Log2 fold change Log2 fold change Log2 fold change (A)-(H) (A)-(M) (H)-(M) Enzyme Candidatesa [average Sigb] [average Sigb] [average Sigb] acetyl/propionyl-CoA carboxylase msed_0147 3.1 [15.9] 3.5 [20.9] 0.4 [1.1] msed_0148 2.8 [15.6] 3.5 [22.0] 0.6 [ 2.3] msed_1375 2.0 [ 3.9] 1.4 [2.7] -0.6 [ 0.8] malonyl-/succinyl-CoA reductase msed_0709 4.6 [17.7] 3.4 [14.0] -1.3 [3.6] 3-hydroxypropionate synthetase msed_1456 2.7 [9.4] 1.8 [ 6.2] -0.9 [2.0] 3-hydroxypropionyl-CoA dehydratase msed_2001 0.6 [1.0] 0.1 [0.1] -0.5 [1.1] acryloyl-CoA reductase msed_1426 1.1 [3.9] 0.3 [1.4] -0.8 [2.8] methylmalonyl-CoA epimerase msed_0639 0.6 [1.1] 0.1 [0.4] -0.5 [0.9] methylmalonyl-CoA mutase msed_0638 2.2 [4.8] 1.5 [3.3] -0.7 [1.1] msed_2055 + msed_2056 1.3 [3.9] + 1.4[2.9] 1.4 [5.1] + 1.3[3.1] 0.1 [0.1] + -0.1[0.3] succinate semialdehyde reductase msed_1424 1.3 [3.8] 1.1 [3.3] -0.2 [0.7] 4-hydroxybutyryl-CoA synthetase msed_1422 3.0 [11.0] 2.6 [10.6] -0.4 [0.8] 4-hydroxybutyryl-CoA dehydratase (2 candidates) msed_1220 -0.7 [1.7] -0.2 [0.3] 0.5 [1.4] msed_1321 2.9 [9.5] 3.2 [12.3] 0.2 [0.4] Crotonyl-hydratase (5 candidates) msed_0336 0.5 [1.6] 0.4 [1.3] -0.1 [0.4] msed_0384 -1.5 [5.1] -1.9 [9.2] -0.5 [1.2] msed_0385 -1.3[3.1] -1.9 [6.6] -0.6 [1.3] msed_0399 -0.1 [0.2] 0.0 [0.2] 0.1 [0.3] msed_0566 0.2 [0.6] 0.2 [0.3] 0.0 [0.6] 3-hydroxybutyryl-CoA dehydrogenase (3 candidates) msed_0389 0.6 [1.1] 0.4 [0.8] -0.2 [0.3] msed_1423 0.8 [1.8] 0.8 [2.2] 0.0 [0.6] msed_1993 + msed_1994 2.0 [6.5] + 1.9[5.6] 1.5 [4.9] + 1.6[5.0] -0.5 [1.0] + -0.3[0.6] acetoacetyl-CoA β-ketothiolase (7 candidates) msed_0270 -0.9 [1.7] 0.7 [1.6] 1.6 [5.3] msed_0271 -0.7 [0.9] 0.5 [0.6] 1.2 [2.4] msed_0386 1.1 [0.9] 1.0 [0.8] -0.1 [0.5] msed_0396 -0.2 [0.3] -0.6 [1.2] -0.4[0.3] msed_0656 1.6 [4.8] 1.4 [4.6] -0.2 [0.4] msed_1290 -2.0 [5.9] -2.5 [9.5] -0.5 [0.9] msed_1647 -0.8 [0.8] -0.8 [1.0] 0.0 [0.2] a Taken from Berg et al 2007, with the exception of msed_2056 and msed_1994 which are suggested by this work. b Significance = -log10(p-value). For reference, a significance value of 5.4 correlates to a p-value of 4 x 10-6.

157

Table 4.A5: Potential ORFs involved with NiFe hydrogenases in M. sedula. Significant differential transcription is considered to have occurred where log2 fold change has a magnitude greater than 1 (equivalent to a 2-fold change) with significance at or above the Bonferroni (≥5.4 for this data set). Average of log2 fold changes and significance values are reported for all 4 combinations of biological repeats (i.e. average of log2 fold changes for autotrophy vs heterotrophy averages values for individual comparisons A1-H1, A1-H2, A2-H1, and A2-H2) Average Average Log2 fold Average Log2 fold change Log2 fold change change (A)-(H) (A)-(M) (H)-(M) M. sedula ORFs [average Siga] [average Siga] [average Siga] putative membrane-bound hydrogenase msed_0944 (β) 3.7 [12.5] 0.9 [2.0] -2.7 [9.9] msed_0945 (α) 2.9 [14.6] 0.9 [3.3] -2.0 [10.5] msed_0946 (anchor) 3.4 [10.1] 2.1 [5.8] -1.3 [3.1] Fe-S oxidoreductase-like protein msed_0947 2.0 [9.1] 1.1 [4.6] -0.9 [3.6] associated hypothetical proteins msed_0948 3.6 [14.5] 1.5 [4.8] -2.2 [8.3] msed_0949 4.1 [18.7] 3.0 [14.7] -1.1 [4.0] msed_0950 1.8 [5.9] 1.1 [3.0] -0.8 [1.8] putative soluble hydrogenase msed_0923 (α) 0.2 [0.3] 0.2 [0.4] 0.0 [0.1] msed_0924 (β) 1.0 [4.0] 0.3 [1.0] -0.7 [3.2] associated hypothetical proteins msed_0921 -0.4 [0.4] -0.3 [0.3] 0.1 [0.2] msed_0922 0.8 [2.1] 0.3 [0.6] -0.5 [1.5] putative accessory proteins msed_0913 (hypA) 1.1 [2.3] 0.5 [0.8] -0.6 [1.2] msed_0915 (hypF) 0.5 [1.5] -0.2 [1.0] -0.7 [2.3] msed_0916 (maturation protease) 0.0 [0.2] -0.2 [0.5] -0.2 [0.6] msed_0917 (hypC) 0.7 [1.5] 0.7 [1.8] 0.1 [0.7] msed_0918 (hypC2) 2.1 [4.7] -0.4 [0.6] -2.5 [7.4] msed_0930 (hypD) 2.5 [5.7] 1.7 [3.9] -0.7 [1.2] msed_0931 (hypC3) 3.0 [8.0] 2.0 [5.2] -1.0 [1.9] msed_0933 (hypE) 2.0 [5.7] 1.8 [5.7] -0.2 [0.3] Other related proteins msed_0914 0.9 [2.3] 0.9 [2.3] 0.0 [0.7] (similarities to cytosolic Fe-S cluster assembly proteins) msed_0919 0.8 [2.1] -0.2 [0.4] -1.0 [3.5] (pqqE-like protein with CX3CX2C and CX2CX3C FeS cluster-binding motifs)

158

(Table 4.A5 continued)

Other gene neighborhood proteins msed_0920 (pseudo-NiFe-α ) 0.9 [2.7] 1.2 [4.4] 0.3 [1.8] msed_0925 (hypothetical) 0.3 [0.4] 0.1 [0.1] -0.2 [0.3] msed_0926 (HTH regulator) 0.3 [1.6] -0.7 [3.7] -1.0 [5.7] msed_0927 (hypothetical) 0.8 [1.1] 0.6 [0.9] -0.2 [0.2] msed_0928 (hypothetical) 1.4 [4.1] 1.0 [3.1] -0.4 [0.8] msed_0929 (adducin/aldolase) 0.9 [3.1] 0.2 [0.2] -0.8 [2.5] msed_0932 (hypothetical) 3.4 [14.1] 1.7 [6.6] -1.7 [6.5] msed_0934 (hypothetical) 1.5 [3.4] 0.6 [1.4] -1.0 [2.0] msed_0935 (aa permease-like) 3.5 [17.9] 1.7 [8.3] -1.8 [8.7] msed_0936 (hypothetical) 1.0 [4.0] 0.7 [3.3] -0.2 [0.8] msed_0937 (hypothetical) 2.2 [4.3] 0.3 [0.4] -1.8 [3.9] msed_0938 (hypothetical) 1.2 [3.6] -0.6 [1.7] -1.9[ 7.8] msed_0939 (thioredoxin domain) 1.0 [2.0] 1.0 [2.4] 0.0 [0.3] msed_0940 (hypothetical) 2.1 [13.6] 1.4 [8.9] -0.8 [4.6] msed_0941 (abortive infection) 0.3 [0.5] -0.1 [0.2] -0.4 [0.8] msed_0942 (hypothetical) 0.7 [1.8] 0.2 [0.6] -0.5 [1.2] msed_0943 (hypothetical) 1.1 [2.6] 1.1 [2.8] 0.0 [0.6] a Significance = -log10(p-value). For reference, a significance value of 5.4 correlates to a p-value of 4 x 10-6.

159

Table 4.A6: Potential ORFs involved with electron transfer from hydrogen and/or sulfur in M. sedula. Significant differential transcription is considered to have occurred where log2 fold change has a magnitude greater than 1 (equivalent to a 2-fold change) with significance at or above the Bonferroni (≥5.4 for this data set). Average of log2 fold changes and significance values are reported for all 4 combinations of biological repeats (i.e. average of log2 fold changes for autotrophy vs heterotrophy averages values for individual comparisons A1-H1, A1-H2, A2-H1, and A2-H2) Average Average Average Log2 fold change Log2 fold change Log2 fold change (A)-(H) (A)-(M) (H)-(M) M. sedula ORFs [average Siga] [average Siga] [average Siga] Sox terminal oxidase cluster msed_0285 (soxD) 1.7 [5.9] -0.2 [0.3] -1.9 [8.2] msed_0286 (soxD') 1.3 [4.5] -0.4 [1.0] -1.7 [8.1] msed_0287 (hypothet.) 1.5 [3.1] 0.1 [0.1] -1.4 [3.4] msed_0288 (soxL) 1.3 [1.9] 0.0 [0.4] -1.4 [2.3] msed_0289 (soxA) 1.0 [2.7] 0.0 [0.2] -1.0 [3.3] msed_0290 (soxB) 1.2 [4.1] 0.5 [1.5] -0.7 [2.2] msed_0291 (soxC) 1.5 [2.8] 0.6 [1.1] -0.9 [1.4] msed_0292 (DUF1404) 1.8 [6.2] 1.1 [3.7] -0.6 [1.6] Heterodisulfide reductase-like cluster msed_1542 (hdrB1-like) 1.0 [1.9] 1.5 [4.0] 0.5 [0.7] msed_1543 (hdrC1-like) 1.3 [3.4] 1.8 [6.1] 0.4 [1.2] msed_1544 0.9 [1.7] 1.8 [5.3] 0.9 [1.8] msed_1545 (hdrA-like) 1.2 [2.2] 1.8 [4.9] 0.6 [0.9] msed_1546 (hdrB2-like) 1.3 [3.3] 1.2 [3.4] -0.1 [0.8] msed_1547 (hdrC2-like) 0.7 [1.4] -0.3 [0.6] -1.0 [2.6] msed_1548 (regulator?) 0.8 [1.7] -0.3 [0.5] -1.1 [3.2] msed_1549 1.3 [2.9] 0.2 [0.3] -1.0 [2.6] msed_1550 1.0 [1.6] 1.7 [4.5] 0.7 [1.2] Sulfur reductase-like cluster msed_0810 (regulator?) 0.6 [1.3] 0.7 [1.9] 0.2 [0.5] msed_0811 0.2 [0.2] 0.7 [1.2] 0.5 [0.8] msed_0812 (mem. anchor?) -0.1 [0.3] 0.3 [0.5] 0.3 [0.4] msed_0813 (TPR repeat domains) 0.5 [1.5] 0.1 [0.3] -0.5 [1.3] msed_0814 (dmsA-like) 0.4 [0.6] 1.1 [2.0] 0.7 [1.2] msed_0815 (sreB-like ferredoxin) 0.3 [0.6] 0.3 [1.0] 0.1 [0.4] msed_0816 -0.3 [1.8] 0.2 [0.7] 0.5 [2.9] msed_0817 (sreD-like ferredoxin) -0.9 [2.5] 0.0 [0.9] 0.9 [2.6] msed_0818 0.1 [3.0] -0.4 [0.8] -0.5 [1.2] a Significance = -log10(p-value). For reference, a significance value of 5.4 correlates to a p-value of 4 x 10-6.

160

Table 4.A7: Potential hydrogenase ORFs involved with electron transfer from sulfur in M. sedula. Significant differential transcription is considered to have occurred where log2 fold change has a magnitude greater than 1 (equivalent to a 2-fold change) with significance at or above the Bonferroni (≥5.4 for this data set). Transcription data taken from NCBI GEO GSE12044. No source of exogenous H2 was provided for these experiments {Auernik and Kelly, 2008}. Y = yeast extract, YS = yeast extract + elemental sulfur, YFS = yeast extract + ferrous sulfate, YKT = yeast extract + potassium tetrathionate, YKS = yeast extract + potassium sulfate Log2 fold Log2 fold Log2 fold Log2 fold change change change change (Y)-(YS) (YFS)-(YS) (YKT)-(YS) (YKS)-(YS) M. sedula ORFs [Siga] [Siga] [Siga] [Siga] putative membrane-bound hydrogenase msed_0944 (β) -1.4 [10.5] -1.1 [7.7] -1.6 [12.2] -1.4 [11.0] msed_0945 (α) 0.1 [0.5] 0.4 [1.7] 0.1 [0.2] 0.3 [1.2] msed_0946 -1.3 [9.0] -0.8 [4.3] -0.9 [5.8] -1.2 [6.9] Fe-S oxidoreductase-like protein msed_0947 0.6 [5.9] 1.1 [10.5] 0.8 [8.7] 1.0 [10.0] a Significance = -log10(p-value). For reference, a significance value of 5.4 correlates to a p-value of 4 x 10-6.

161

Figure 4.A1: Alignment (using ClustalW2) of NiFe hydrogenase alpha subunits highlighting 4 Cys Ni-binding motifs.

162

CLUSTAL 2.0.10 multiple sequence alignment Canonical Cys other Cys

EchE_Mbarkeri_A0148 ------MTTVIPFGPQHPVLPEPVSLKLE 23 mbhL_12__Pfuriosus_PF1434 ------MKKVEYWVKIPFGPIHPGLEEPEKFIIT 28 HycE_Ecoli 150 DSMDYRQRPAPTTDAETYEFINELGDKKNNVVPIGPLHVTSDEPGHFRLF 200 EhbN_Mjannaschii_MJ1027 ------MLFMKYEGEIAIGPVHPTMLEPHRLRLF 28 HycE-like_Msed1429 7 GPYCLTSDGLREGPCIKDQSEEESLGYGSFKFVYGPSAGGLLESVGFEIT 57 HycE-like_Sso1023 18 GDYCLYEKTITEEKCEENKPN-ITQTYGSFKFIYGPSAGGLLETIKFIIT 67 HycE_Pto0826 17 GDLTIYARTYNYDDIEHENEK---RTDFSFNFSYGPLTGGIPEPFRFDIN 64 HycE-like_Tvn1456 17 EKYSVYEHILSDQRREIPSES-YRNLPGEFTFNYGPATGGLIESVAFDFH 66 HynL_Msed_0945 ------MASSSPYYFTFDWTKPVLIEPIVRIKPELGIQVTY 35 HynL-like_SislU328contig56_239 ------MASS--YLFKYDWTKPVVIEPIVRIKPELGIQVSV 33 HynL2_Msed_0923 ------MNEKVISPLNRVEGDLDLKVVF 22 HynL2-like_SislU328contig56_23 ------LTSKIISPFNRVEGDLDVEVKF 22 HynL3_Msed_0920 ------MLNPLALDT--IDLKVKI 16 HynL3-like_SislU328contig56_23 ------MSLYDLEI--IDLDVNI 15 HynL_Aambivalens ------MSNPLTMKIDPITRVAGHLGLTATV 25

EchE_Mbarkeri_A0148 IDDNV------VVGVLPSLGYVHRGLETFINT-KDFNQTTYVCERICGIC 66 mbhL_12__Pfuriosus_PF1434 LDGER------IVNVDVKLGYNLRGVQWIGMR-RNYVQIMYLAERMCGIC 71 HycE_Ecoli VDGEN------IIDADYRLFYVHRGMEKLAETRMGYNEVTFLSDRVCGIC 244 EhbN_Mjannaschii_MJ1027 IEDEI------IKEAELVIGVNYRGIELIMEG-LPPEKISILSEKICGIC 71 HycE-like_Msed1429 TYGES------IEKINHLPY-KGRQITLSGLT---IGDALLRVERINGAF 97 HycE-like_Sso1023 TNGEK------ILGIDAEVY-KNREIVISGLT---VDDALLRVERINAPF 107 HycE_Pto0826 TSGEK------INYIKYYNY-KIRNIKLKGIN---IKDAIPYIERINGNF 104 HycE-like_Tvn1456 TPGEL------IKHVDVYPYIKTRKIKVRGLS---PEDALLLVERINGFH 107 HynL_Msed_0945 DSSSN------RVTDAYAAAGMFRGFEIFMRN-KPGPDVIMLSSRECGIC 78 HynL-like_SislU328contig56_239 NIEGQNQ---VRAVEAYAGAGMFRGFEIFMRN-KPAPDVIMLSSRECGIC 79 HynL2_Msed_0923 EG-KK------VVKAFP-MSRLFRGIEIILKG-KFPMDSLVITPRICGIC 63 HynL2-like_SislU328contig56_23 KE-NK------VVDAKI-VSRLFRGLELILRG-KSPLDSLVITPRVCGIC 63 HynL3_Msed_0920 SD-GK------VVYAKS-SGNKLRGYERMFIG-KDPRELPDIIPRVLSTC 57 HynL3-like_SislU328contig56_23 RD-KK------VVSAKC-SGNKVRGFEKAFTG-KDAKTLYEVLPRVLATC 56 HynL_Aambivalens DPNTG----QVINNQAYTYVTMFRGFEVFLRG-RQPPDAVHIVSRSCGVC 70 * .

163

EchE_Mbarkeri_A0148 345------VLTIDPCVSCTER------358 mbhL_12__Pfuriosus_PF1434 367------IASIDPCLSCTDRVAIVKEGKKVVLTEKDLLKLSI 402 HycE_Ecoli 524------IGSLDPCYSCTDRMTVVDVRKKKSKVVPYKELERY 559 EhbN_Mjannaschii_MJ1027 355------IASCDPCFTCTDRMIVVKENKI------377 HycE-like_Msed1429 352------WESFGIWISELEVDLE------368 HycE-like_Sso1023 375------WESFGIWVSEIGVMLK------391 HycE_Pto0826 366------FESFGLWAADEVNII------381 HycE-like_Tvn1456 373------WESFGIWVSELAVVIQ------389 HynL_Msed_0945 690------DGFEFAQTLRSFDPCFVCGVHMVLPNGRARYITLGAPVDLSNA 733 HynL-like_SislU328contig56_239 697------DGFEFASTLRSFDPCFVCGVHVVLPNGKVRYITLGAPIDLSES 740 HynL2_Msed_0923 480------PIEVAHIVRSHDACMVCNVHVLDG--GKEILSMRL------513 HynL2-like_SislU328contig56_23 478------PIEVYHIVRSHDPCMVCNVHML-----KLI------503 HynL3_Msed_0920 342------PWEISLAVSSLDSCFITNVEVYLDDKYLFTKRVGGFC----- 379 HynL3-like_SislU328contig56_23 351------PVELALTVSSLDTCFVTRVSVFENGKLVTTKRIGGFC----- 388 HynL_Aambivalens 589------YDFLRAIRSFDPCLVCAAHFEIKGKNKKLEHIITPVCNS-- 628

164

Figure 4.A2: Phylogram (using ClustalW2 alignment) of NiFe hydrogenase beta subunits

165

Chapter 5

Bioleaching transcriptomes of Metallosphaera sedula reveal complex mixotrophic physiology

Kathryne S. Auernik and Robert M. Kelly

Department of Chemical and Biomolecular Engineering North Carolina State University Raleigh, NC 27695-7905

Submitted to Applied and Environmental Microbiology (April 2009)

166

ABSTRACT

The extremely thermoacidophilic crenarchaeon Metallosphaera sedula, grown autotrophically on (H2+CO2) was inoculated into cultures containing 10g/L chalcopyrite

(CuFeS2). Once active bioleaching was confirmed (relative to chemical leaching observed in abiotic controls), cultures were harvested and RNA was extracted to compare the chalcopyrite transcriptome(s) against autotrophic growth in the absence of the metal sulfide.

Transcriptomics revealed chalcopyrite stimulation of a rusticyanin-like blue copper protein transcript (Msed_0966) reported to be important for iron oxidation in the well-studied bioleacher, Acidithiobacillus ferrooxidans, a putative sulfide: quinone oxidoreductase

(Msed_1039), a doxDA thiosulfate: quinone oxidoreductase (TQO) implicated in reduced inorganic sulfur compound (RISC) oxidation, and the doxBCE terminal oxidase originally characterized in sulfur-oxidizing Acidianus ambivalens. Differential transcription observed in foxAA’ (Msed_0484/Msed_0485) is discussed with respect to its role in iron oxidation in

M. sedula and the obligate autotrophic bioleacher S. metallicus. A down-regulation of the 3- hydroxypropionate/4-hydroxybutyrate cycle for inorganic carbon fixation after 9 days on

CuFeS2 suggests a switch to mixotrophic growth, most likely via recycling of lysed cellular material. Additional support for mixotrophic growth on CuFes2, provided by transcriptional patterns of ORFs previously suggested to be indicators of heterotrophy, autotrophic biosynthesis, and mixotrophy, is presented and discussed.

167

INTRODUCTION

With dwindling high-purity mineral deposits and increasing environmental concerns, an increase in bioleaching operations in the mining community would offer the opportunity for product recovery from lower grade ores. Although, in the past, lower commodity prices combined with significant recovery costs have limited economic feasibility of specific operations (30), a variety of acidophilic bacteria and archaea have been widely studied for their role in bioleaching processes (30, 33, 35). While the majority of the bacteria studied have been mesophilic, there has been increased interest in moderately or extremely thermoacidophilic organisms recognized for their ability to catalyze the bioleaching process at elevated temperatures favoring reaction kinetics (and therefore decreased processing times)(14, 15, 20). This is especially relevant for chalcopyrite (CuFeS2) bioleaching, as higher temperatures can eliminate passivation which slows leaching rates at the mineral surface(29, 30). Extreme thermoacidophiles which have demonstrated leaching capabilities include Sulfolobus species, Acidianus species, and Metallosphaera species (8, 11, 14, 19,

28, 29, 31). Recent work with bioleaching organisms suggest that use of a consortia is the most effective approach, presumably because one species has limitations that can be relieved by the capabilities of another (29, 34).

M. sedula is an extremely thermoacidophilic bioleaching crenarchaeon of the

Sulfolobales order which was recently genome sequenced(4). Using a functional genomics approach, combining comparative genomics and transcriptomics, ORFs related to iron oxidation and sulfur oxidation have been revealed and specific components involved with transfer of electrons from these inorganic energy sources have been highlighted (3).

168

Biochemical and genomic work with autotrophically grown M. sedula has revealed a new cycle for inorganic carbon fixation (1, 2, 10, 23) which elucidates a survival mechanism important in conditions where organic carbon may be limiting. The functional genomics approach has also been used to examine the components that influence M. sedula’s ability to survive in idealized heterotrophic, autotrophic, and mixotrophic conditions where organic carbon/energy sources are available in abundance {Auernik and Kelly, submitted}. Here,

M. sedula growth on chalcopyrite was examined using transcriptomics to determine physiological growth factors related to mineral sulfide bioleaching.

MATERIALS AND METHODS

Growth of M. sedula cultures. M. sedula (DSMZ 5348) was grown aerobically at

70°C in an orbital shaking oil bath at 70 rpm on DSMZ 88 medium (pH 2). Autotrophically grown cultures (headspace content: 7% CO2, 14% O2, 28% H2, with the balance N2) were used to inoculate 1L bottles containing 300mLs of media 88, supplemented with 10g/L chalcopyrite (provided by Greg Olsen), and a headspace containing either air, air enhanced with CO2 (7% final concentration), or the autotrophic mix mentioned above. Use of inoculum from autotrophic cultures created initial cell densities ranging from 106 to 107 cells/mL. Cell densities were measured using epifluorescence microscopy. Autotrophic cultures were harvested during exponential phase growth (1 day post-inoculation) to represent inoculum, and chalcopyrite cultures were harvested at various points in stationary phase (3, 6, or 9 days post-inoculation). Harvest occurred by quickly chilling cultures and then centrifuging at

9,510 x g for 15 min at 4°C.

169

M. sedula oligonucleotide microarray construction and transcriptional response analysis. A spotted whole-genome oligonucleotide microarray based on at least

2,256 protein-coding open reading frames (ORFs) was constructed (2,328 probes with 5 replicates of each, yielding 11,640 spots per array), as described previously (3, 4). Probes

(60-mers) were designed using OligoArray, version 2.1, software (36) and synthesized by

Integrated DNA Technologies (Coralville, IA). RNA was extracted (Trizol; Invitrogen) and purified (RNeasy Mini Kit; Qiagen), reverse transcribed (Superscript III; Invitrogen), repurified, labeled with either Cy3 or Cy5 dye (GE Healthcare), and hybridized to one of eight microarray slides (Corning). Slides were scanned on a Packard BioChip Scanarray

4000 scanner, and raw intensities were quantitated using ScanArray Express, version

2.1.8, software. Normalization of data and statistical analysis were performed using JMP

Genomics, version 3.1, software (SAS, Cary, NC). In general, significant differential transcription, or “response,” was defined to be relative changes at or above 2 (where a log2 value of ±1 means a twofold change) with significance values at or above the Bonferroni correction, which was 5.4 (equivalent to a P value of 4.0 x 10- 6) for this data set.

Comparative genomics analysis. For comparative genomics, M. sedula gene sequences and annotations were downloaded from the Joint Genome Institute (JGI) microbial genome website (http://genome.ornl.gov/microbial/msed/28feb07/) or GenBank.

Other sequences referenced were obtained from the GenBank. The Basic Local Alignment

Search Tool (BLAST) searches were conducted using a BLOSUM62 matrix with either the

NCBI protein-protein blast program (BLASTP) against the Swissprot database, the

170 nonredundant database, or against the specific sequenced genomes of interest (in either

GenBank or JGI/Oak Ridge National Laboratory databases).

Iron Assay. The amount of soluble iron and its oxidation state in M. sedula cultures was tracked using an o-phenanthroline colorimetric assay modified from ASTM E3934 and

(26). In brief, 25uL of culture supernatant (diluted as needed to keep total iron content <

5ug) was added to 1.118mL of deionized water. Replicates used to evaluate total iron were treated with reducing agent hydroxalamine hydrochloride (25uL of a 10% solution), while replicates evaluating ferrous iron content were treated with 25uL of deionized water

(control). Phenanthroline reagent was added (63uL of a 0.3% solution), followed by ammonium acetate buffer (20uL, pH 4.5, made with 25g of ammonium acetate dissolved in

15mL deionized water and mixed with 70mL of glacial acetic acid), and the reddish orange color was allowed to develop at room temperature for 15 minutes out of direct sunlight before aliquotting in triplicate on a 96-well plate and measuring absorbances at 520nm.

Calibration curves, created using serial dilutions of a freshly prepared FeSO4 solution ranging from 0-200mg/L Fe(II), were processed as total iron samples and run on every plate in triplicate. Ferric iron content was calculated as the difference between total and ferrous iron content. Use of concentrated phenanthroline solutions recommended by (26) to alleviate Cu interference was not necessary, as copper levels in the final test solutions did not exceed 10ppm.

Microarray data accession number. Raw data, as well as final log2 relative changes and significance values (where significance = -log10(p-value)), will be deposited in

171 the NCBI Gene Expression Omnibus (GEO) database upon submission of this manuscript for journal publication. Any M. sedula microarray data referenced from previous studies can be found in GEO under series accession numbers GSE14978 (heterotrophy, autotrophy, mixotrophy data [H, A, M conditions]), GSE12044 (iron and sulfur compound data [Y, YFS,

YKS, YKT, and YS conditions]), or GSE11296 (FeSO4 effect data [YE, YEF conditions]).

RESULTS AND DISCUSSION

Growth physiology. M. sedula cultures were grown autotrophically utilizing 3 different headspace compositions: 100% air, 7% CO2 + balance air, 7% CO2 + 14% H2 + balance air. Significant differences in cell growth rates, cell densities, and iron release rates were observed for the 3 conditions. Figure 1 depicts growth curves for M. sedula cells for each condition tested. Cultures supplemented with H2 + CO2 exhibited faster growth rates than cultures supplemented with CO2 or cultures with no added headspace supplements

(“air-only”), reaching maximum density earlier than other cultures. This suggested that the added H2 was serving as an energy or reductant source for M. sedula in the presence of chalcopyrite. To verify that active bioleaching was occurring in the cultures, levels of iron in inoculated cultures were tracked against abiotic controls containing the same headspace supplements. Total iron released is reported for all conditions, as virtually all the soluble iron was present in the reduced Fe2+ state. Iron present in solution at time = 0 hours is a result of chemical leaching of chalcopyrite at 70oC in DSM 88 for 3 days (6 days in the case of CO2 enhanced cultures) prior to inoculation to ensure immediate availability of soluble

172 iron as an energy source at the time of inoculation. Biotic iron release (bioleaching) in the

H2+CO2 supplemented cultures was clearly evident at 6 and 9 days post inoculation, suggesting that iron is serving as an energy source, in addition to hydrogen. Interestingly, a biological effect is visible as early as day 3 in H2 + CO2 and air-only cultures, as soluble iron levels are less than the abiotic controls. While Figure 1 iron assay data regarding active bioleaching in air-only and CO2 enhanced cultures is arguably inconclusive, visual inspection of culture bottles (Figure 2) showed formation of rust-colored precipitate on glass walls, strongly suggesting that bioleaching was occurring at some point. Following harvest of CO2-enhanced cultures, culture bottles were rinsed and dried to remove all residual media. Precipitate from walls was dissolved using 10N HCl, the pH and working volume were restored to that of the initial culture, and now-soluble total iron levels were determined to be approximately 85mg/L. Combined with the soluble iron levels at the time of harvest (31mg/L), the iron released from CO2 enhanced cultures was twice that of its abiotic control (116mg/L vs 54mg/L). This supports the idea that active bioleaching had occurred in this culture, although the recovery of precipitate after the fact makes it somewhat difficult to estimate at what point bioleaching begins to occur above abiotic levels. For this reason, RNA from H2 + CO2 supplemented chalcopyrite cultures was extracted at 3 (“d3”) and 9 (“d9”) days post-inoculation (3 cultures harvested for each time point) and compared to RNA from 4 actively growing H2+CO2 cultures in the absence of chalcopyrite (representative of initial inoculum used, “d0”) to examine the impact of bioleaching on the M. sedula transcriptome.

173

Iron oxidation. As previously discussed (4), the blue copper protein rusticyanin

(Rus) from A. ferrooxidans is also believed to be important for electron transfer from inorganic electron donors, primarily Fe(II). It has been characterized by its high redox potential (+600mV), twice the value typical of type 1 copper proteins (7). In A. ferrooxidans,

Rus is proposed to serve as a periplasmic intermediate electron acceptor from an outer membrane cytochrome c which donates electrons either to a downhill respiratory pathway or an uphill pathway in support of reducing equivalent generation (13). Of the 2 putative rusticyanin candidates in M. sedula, Msed_0966 was observed to be up-regulated 6.7-fold

(p-value 4.2 x 10-5) and 10.5-fold (p-value 2.3 x 10-6) 3 and 9 days following inoculation on chalcopyrite compared to the autotrophic growth in the absence of chalcopyrite (d0)

(Figure 3, Table S1). According to SignalP 3.0 trained on gram-positive bacterial sequences(9), a signal peptide is predicted with low probability, while a new model, PRED-

SIGNAL, trained on archaeal sequences (5), suggests only a N-terminal transmembrane helix, so it is not clear whether Msed_0966 is more likely to serve as an initial electron acceptor associated with the outside of the membrane, or as an intermediate electron acceptor/donor associated with the inner membrane.

It has also been postulated that the Sulfolobus cytochrome b (CbsAB) may be involved with transfer of electrons from external inorganic donors such as Fe(II). Two loci within the M. sedula genome contain CbsAB-like ORFs: the SoxNL-CbsAB cluster

(Msed_0500-Msed_0504) described in S. acidocaldarius (22), and the FoxAA’BCD cluster

(Msed_0468-Msed0485) originally described in S. metallicus (8). While the redox potential of Fox protein components has not been measured, work with the SoxNL-CbsAB in S. acidocaldarius shows that similar to blue copper proteins, redox potentials are high,

174 especially for the CbsA proteins (+400mV) (21). Recent work in A. ambivalens proposes that electron transfer proceeds analogous to a bc1 complex mechanism, i.e. SoxN

(+160mV, +200mV) to SoxL (+320mV) to CbsA (+400mV)(6). While findings of A. ambivalens’ SoxN glycosylation support this protein as an outer membrane/initial electron acceptor, CbsA was reported to be the glycosylated protein in S. acidocaldarius, as is predicted by the S/T-rich C-terminal domain of CbsA (Msed_0504) in M. sedula. soxNL- cbsABA’ was upregulated in M. sedula on chalcopyrite (Figure 3, Table S2) consistent with with previous microarray data suggesting upregulation on Fe(II) supplements (3, 4).

The other set of CbsAB-like sequences in M. sedula include FoxCD

(Msed_0478/Msed_0477) which were originally described as being important for iron oxidation in the obligate autotroph, S. metallicus (8). Somewhat surprisingly, components of the fox cluster (including foxC) had lower transcription in the presence of iron compared to its absence when studied in M. sedula, although transcription levels were relatively high on iron (4). After longer exposure to iron, it was noticed that maximum transcripts of foxA

(Msed_0484) appeared to correlate with minimum transcript levels of foxA’ (Msed_0485)

(3). Performance on chalcopyrite revealed a similar response, with foxA up-regulated 3.0- fold (d3 vs d0, p-value 1.8 x 10-7) and foxA’ down-regulated 5.1-fold (d9 vs d0, p-value: 2.5 x 10-6) (Figure 3, Table S2). Downregulation of foxCD was also observed on chalcopyrite compared to autotrophic growth in the absence of Fe(II). This suggests that while foxA may allow the M. sedula fox cluster to function similar to the S. metallicus fox cluster in the presence of Fe(II), the M. sedula fox cluster may also play an important role in electron transfer involving non-metal electron donors (inorganic or organic). Both foxA and foxA’ encode proteins with similarity to the SoxB/CoxI-like terminal/cytochrome oxidase subunits

175 involved with proton-pumping that contribute to ATP generation through the maintenance of proton motive force (IPR000883).

Just downstream of the SoxNL-cbsAB cluster lies approximately a dozen additional

ORFs also up-regulated on chalcopyrite (Table S3). Msed_0507-Msed_0510 appear to encode a pyruvate:ferredoxin oxidoreductase which has been previously suggested as the most likely candidate to function as a pyruvate synthase converting acetyl-CoA generated during inorganic carbon fixation into pyruvate using electrons from a reduced ferredoxin

(Auernik and Kelly, submitted). In addition to upregulation in the presence of Fe(II) (3, 4), this complex is also up-regulated on autotrophy and/or mixotrophy compared to heterotrophy in low-iron systems, suggesting that other inorganic electron sources (i.e. H2) may also stimulate transcription. Msed_0511-Msed_0518 possess only general annotations: Msed_0511/Msed_0512/Msed_0515 are annotated as radical SAM domain proteins and possess some similarity (24-32% identity) to bacterial PqqE proteins implicated in pyrrolo-quinoline quinone cofactor biosynthesis (17, 18, 25), while hypothetical protein Msed_0518 appears to have similarity to several uncharacterized flavoproteins.

Because of their repeated up-regulation on iron-containing conditions, these ORFs warrant further investigation in relation to electron transfer from inorganic energy sources.

Sulfur oxidation. Several proteins have the potential to be involved with initial electron acceptance from RISCs, including a putative tetrathionate hydrolase (TetH,

Msed_0804), a thiosulfate: quinone oxidoreductase (DoxDA, Msed_0363/Msed_0364) characterized in A. ambivalens (27), and a putative sulfide: quinone oxidoreductase (SQR,

Msed_1039) all previously described in M. sedula (3, 4). No response was seen from any

176 of the ORFs encoding these proteins after 3 days on chalcopyrite (TableS4). By 9 days on chalcopyrite, a 2.0-fold/3.1-fold up-regulation was observed in doxDA (p-values 1.1 x 10-

3/9.8 x 10-9) and a 3.4-fold up-regulation of putative sqr (p-value 6.3 x 10-10), which is predicted to be targeted to the outside of the membrane by both SignalP and the archaeal

PRED-SIGNAL (Figure 3). No up-regulation of tetH was observed on chalcopyrite during this time, which may be influenced to the relative nature and/or abundance of sulfur intermediates produced via the polysulfide mechanism of leaching believed to occur with chalcopyrite (35). The reason for delay in up-regulation is unclear, although it could be related to preferential energy sources or timing of bioenergetic needs. The delay is not seen with other clusters of genes purported to be involved with electron transfer from RISCs.

Up-regulation of parts of the RISC-induced terminal oxidase soxABCDD’L is seen at day 3 on chalcopyrite, with the strongest response in soxDD’ (Figure 3, Table S5).

Transcripts from SoxBC are not differentially transcribed on chalcopyrite compared to the autotrophically grown inoculum. This may be related to the previous observation that H2 also appears to stimulate soxABCDD’L transcription (Auernik and Kelly, submitted).

Interestingly, from day 3 to day 9, a down-regulation of soxAL (2.4-fold/1.6-fold, p-values

9.3 x 10-12/1.6 x 10-19) is observed. This day 9 down regulation is not observed in the other terminal oxidase presumed to be stimulated by RISCs, DoxBCE

(Msed_2032/Msed_2031/Msed_2030), originally characterized in A. ambivalens (32). doxBCE was up-regulated 4.0-fold/2.0-fold/6.8-fold (p-values 2.6 x 10-11/1.1 x 10-10/6.4 x 10-

9) at day 3 on chalcopyrite, with no significant differential transcription between day 3 and 9

(Figure 3, Table S5). Although DoxBCE was originally characterized in a sulfur oxidizing environment in A. ambivalens, doxB was previously observed to be up-regulated in the

177 presence of an Fe(II) substrate (3), so the presence of Fe(II) in this system should also be considered as an influencing factor in M. sedula.

While electrons from RISCs transferred to the proton-pumping terminal oxidases mentioned above support ATP generation, they also potentially contribute to the generation of reducing power. Two clusters of ORFs were previously recognized to be stimulated in the presence of RISCs in M. sedula: the Dms/Sre-like cluster (Msed_0810-Msed_0818) and the

Hdr-like cluster (Msed_1542-Msed1550) (3). The Hdr-like cluster, but not the Dms/Sre-like cluster, has also been observed to be stimulated by H2 (Auernik and Kelly, submitted).

Msed_0815 and Msed_0817 contain ferredoxin domains, while Msed_1545 encodes an

HdrA-like pyridine-nucleotide disulfide oxidoreductase with a NAD/FAD-binding region at its

N-terminal. Although Msed_1545 was not transcribed significantly higher in the presence of chalcopyrite added to H2 already present in the inoculum, both ferredoxin domain proteins were upregulated after 3 days on chalcopyrite (Figure 3, Table S6). Components of both clusters exhibited a down-regulation of transcription from day 3 to day 9 similar to that mentioned for the SoxABCDD’L complex. Reasons for this day 3 → day 9 down-regulation will be discussed later.

Metal Tolerance. One well characterized mechanism of metal tolerance involves the CopMA Cu+-efflux ATPase characterized in S. solfataricus(16). Both copMA

(Msed_0490/Msed_0491) were up-regulated ≥ 5.5-fold (p-values ≤ 2.1 x 10-10) in the presence of chalcopyrite at 3 and 9 days. There was no differential transcription of the copT regulator, consistent with constitutive transcription observed in S. solfataricus. Although there was no known mercury in the system, a transient increase in merAH

178

(Msed_1241/Msed_1242) was noted on day 3 (3.1-fold/19.3-fold increase over d0 with p- values of 2 x 10-11/1.1 x 10-13), with day 9 transcript levels equivalent to d0 levels. Proteins encoded by these two ORFs are predicted to contain similarities to the merAH

(SSO2689/SSO10899) involved with mercury tolerance in S. solfataricus (37, 38). The closest match to MerR (SSO2688) was Msed_1005 (57% ID), also up-regulated 6.1-fold on chalcopyrite at day 3 (p-value 1.0 x 10-5). Interestingly, the regulator also remained upregulated at day 9 (3.6-fold vs day 0, p-value 6.0 x 10-4). The behavior of the mercuric reductase and associated regulator transcripts raise the question as to whether heavy metal-tolerating activity extends beyond mercury in M. sedula.

Growth mode. Although it is clear that the exponential phase inoculum used for initiation of chalcopyrite cultures was growing autotrophically on inorganic carbon and H2, it is unclear what growth mode exists during growth on chalcopyrite. Clearly multiple forms of inorganic energy sources are available, as chalcopyrite provides access to Fe(II) and

RISCs, as well as the H2 already present. However, at longer culture times, when cell densities fail to increase further, it is assumed that cell growth rate is equal to the death rate, and a potential source of organic carbon (and possibly energy sources) becomes available via lysed cells. Enzymes functioning in the 3-hydroxypropionate/4- hydroxybutyrate cycle of inorganic carbon fixation are highly transcribed at day 0 and day 3, but exhibit a decrease in transcription at day 9 (Figure 4, Table S7). This includes the putative carbonic anhydrase (Msed_0390) which may contribute to interconversion of CO2 to bicarbonate (Table S8). Although malonyl-/succinyl-CoA reductase (Msed_0709) transcriptional patterns are not readily apparent on a heat plot, this ORF was also down-

179 regulated 5.7-fold (p-value 1.5 x 10-15) between day 3 and day 9. Because cell density is relatively steady during this time, and based on previous observation that M. sedula exhibits a strong preference for organic carbon when present (Auernik and Kelly, submitted), this data suggests that M. sedula is utilizing organic carbon for mixotrophic growth by day 9.

Several ORFs whose up-regulation was considered to be characteristic of heterotrophic growth in M. sedula were also seen to be up-regulated as early as day 3, continuing through day 9 (Figure 4, Table S9). This includes a putative succinyl-CoA synthetase

(Msed1581/Msed-1582) proposed to be involved with cycling of TCA intermediates,

Msed_1772-Msed1777 proposed to be involved with ring cleavage and aromatic amino acid catabolism, and a putative glutamate dehydrogenase (Msed_2074) involved with removing excess N from organic carbon substrates. Several ORFs involved with biosynthetic functions typically observed to have high transcriptional levels under autotrophic conditions are also down-regulated by day 9 (Figure 4, TableS10). This includes ORFs encoding enzymes involved with nitrogen- and sulfur-assimilating amino acid biosynthesis, such as glutamine/glutamate synthases (Msed_1612 /Msed1884-Msed_1885) and homoserine kinase/cystathionine gamma synthase (Msed_0672/Msed_0673). Msed_0829-Msed0830, proposed to function in isoleucine biosynthesis are also transcribed at lower levels by day

9. Several ORFs found to be down-regulated in mixotrophic growth conditions in M. sedula were also seen to be down-regulated at day 9 on chalcopyrite (Figure 4, Table S11), such as the transcripts of soxM, part of the SoxM supercomplex involved with proton-pumping, previously shown to be upregulated under heterotrophic growth conditions (24). This lends further support to the idea that organic carbon from lysed cells is being assimilated by surviving M. sedula. Downregulation of proteins associated with the membrane bound

180 hydrogenase responsible for H2 utilization at day 9 also hints at the possibility that organic carbon may also be contributing in some way as an energy source (Figure 4, Table S12).

Even though the membrane anchor of hydrogenase is not shown, it is also down-regulated

3.6-fold (p-value 8.7 x 10-23) between day 3 and day 9. Twelve ribosomal components are noted to have transcription levels (lsms 3.0-5.7) in the top 7% of all ORFs at day 9 (i.e.

Msed_1626, Msed_1634, Msed_1635, and Msed_1637 shown in Figure 4), suggesting that transcription is still continuing at relatively high levels. It may be argued that down- regulation of many ribosomal proteins on chalcopyrite by day 3 (resulting in the relatively slow growth rates observed in Figure 1) may also cause a decrease in transcripts resulting in an apparent down-regulation of specific ORFs; however, no significant down-regulation of ribosomal proteins occurred between day 3 and day 9 (Figure 4), supporting the idea that the other changes at day 9 discussed are an indicator of a shift to mixotrophic growth mode. Interestingly, if the natural environment of M. sedula provides access to organic carbons predominantly through lysed cellular material, this may explain why M. sedula heterotrophic growth is easily demonstrated on peptide, but not carbohydrate-containing substrates.

Summary. This work has shown that M. sedula growth, and bioleaching, of chalcopyrite, is actually a complex process involving multiple inorganic sources of electron donors (Fe(II), RISCs, and H2 for this system). While a chalcopyrite bioleaching system may only be supplied with inorganic carbon and energy supplements, the inoculation of the system indirectly supplies a source of organic carbon (and potentially organic energy) such

181 that upon reaching a sufficient cell density, M. sedula will function in a mixotrophic growth mode, recycling components of lysed cells to provide sources of organic carbon (and possibly organic energy).

ACKNOWLEDGEMENTS

We would like to thank Greg Olsen for providing the chalcopyrite used in this work.

KSA acknowledges support from an NIH T32 Biotechnology Traineeship. RMK acknowledges support from the NSF Biotechnology Program.

REFERENCES

2. Alber, B. E., J. W. Kung, and G. Fuchs. 2008. 3-Hydroxypropionyl-coenzyme A synthetase from Metallosphaera sedula, an enzyme involved in autotrophic CO2 fixation. J. Bacteriol. 190:1383-9.

3. Auernik, K. S., and R. M. Kelly. 2008. Identification of components of electron transport chains in the extremely thermoacidophilic crenarchaeon Metallosphaera sedula through iron and sulfur compound oxidation transcriptomes. Appl. Environ. Microbiol. 74:7723-32.

4. Auernik, K. S., Y. Maezato, P. H. Blum, and R. M. Kelly. 2008. The genome sequence of the metal-mobilizing, extremely thermoacidophilic archaeon Metallosphaera sedula provides insights into bioleaching-associated metabolism. Appl. Environ. Microbiol. 74:682-92.

182

5. Bagos, P. G., K. D. Tsirigos, S. K. Plessas, T. D. Liakopoulos, and S. J. Hamodrakas. 2009. Prediction of signal peptides in archaea. Protein Eng. Des. Sel. 22:27-35.

6. Bandeiras, T. M., P. N. Refojo, S. Todorovic, D. H. Murgida, P. Hildebrandt, C. Bauer, M. M. Pereira, A. Kletzin, and M. Teixeira. 2009. The cytochrome ba complex from the thermoacidophilic crenarchaeote Acidianus ambivalens is an analog of bc(1) complexes. Biochim. Biophys. Acta 1787:37-45.

7. Barrett, M. L., I. Harvey, M. Sundararajan, R. Surendran, J. F. Hall, M. J. Ellis, M. A. Hough, R. W. Strange, I. H. Hillier, and S. S. Hasnain. 2006. Atomic resolution crystal structures, EXAFS, and quantum chemical studies of rusticyanin and its two mutants provide insight into its unusual properties. Biochemistry 45:2927-39.

8. Bathe, S., and P. R. Norris. 2007. Ferrous iron- and sulfur-induced genes in Sulfolobus metallicus. Appl. Environ. Microbiol. 73:2491-7.

9. Bendtsen, J. D., H. Nielsen, G. von Heijne, and S. Brunak. 2004. Improved prediction of signal peptides: SignalP 3.0. J. Mol. Biol. 340:783-95.

10. Berg, I. A., D. Kockelkorn, W. Buckel, and G. Fuchs. 2007. A 3- hydroxypropionate/4-hydroxybutyrate autotrophic carbon dioxide assimilation pathway in Archaea. Science 318:1782-6.

11. Bode, M. L., S. R. Buddoo, S. H. Minnaar, and C. A. du Plessis. 2008. Extraction, isolation and NMR data of the tetraether lipid calditoglycerocaldarchaeol (GDNT) from Sulfolobus metallicus harvested from a bioleaching reactor. Chem. Phys. Lipids 154:94-104.

12. Breitkreutz, B. J., P. Jorgensen, A. Breitkreutz, and M. Tyers. 2001. AFM 4.0: a toolbox for DNA microarray analysis. Genome Biol. 2:SOFTWARE0001.

13. Bruscella, P., C. Appia-Ayme, G. Levican, J. Ratouchniak, E. Jedlicki, D. S. Holmes, and V. Bonnefoy. 2007. Differential expression of two bc1 complexes in the strict acidophilic chemolithoautotrophic bacterium Acidithiobacillus ferrooxidans suggests a model for their respective roles in iron or sulfur oxidation. Microbiology 153:102-10.

183

14. Clark, T. R., F. Baldi, and G. J. Olson. 1993. Coal Depyritization by the Thermophilic Archaeon Metallosphaera sedula. Appl. Environ. Microbiol. 59:2375- 2379.

15. Du Plessis, C. A., J. D. Batty, and D. W. Dew. 2007. Commercial Applications of Thermophile Bioleaching. In D. E. Rawlings and D. B. Johnson (ed.), Biomining. Springer-Verlag, Berlin Heidelberg.

16. Ettema, T. J., A. B. Brinkman, P. P. Lamers, N. G. Kornet, W. M. de Vos, and J. van der Oost. 2006. Molecular characterization of a conserved archaeal copper resistance (cop) gene cluster and its copper-responsive regulator in Sulfolobus solfataricus P2. Microbiology 152:1969-79.

17. Felder, M., A. Gupta, V. Verma, A. Kumar, G. N. Qazi, and J. Cullum. 2000. The pyrroloquinoline quinone synthesis genes of Gluconobacter oxydans. FEMS Microbiol. Lett. 193:231-6.

18. Goosen, N., H. P. Horsman, R. G. Huinen, A. de Groot, and P. van de Putte. 1989. Genes involved in the biosynthesis of PQQ from Acinetobacter calcoaceticus. Antonie Van Leeuwenhoek 56:85-91.

19. Han, C. J., and R. M. Kelly. 1998. Biooxidation capacity of the extremely thermoacidophilic archaeon Metallosphaera sedula under bioenergetic challenge. Biotechnol. Bioeng. 58:617-24.

20. Harvey, T. V. D. M., W. 2002. Presented at the Bio & Hydrometallurgy, Capetown, South Africa.

22. Hiller, A., T. Henninger, G. Schafer, and C. L. Schmidt. 2003. New genes encoding subunits of a cytochrome bc1-analogous complex in the respiratory chain

184

of the hyperthermoacidophilic crenarchaeon Sulfolobus acidocaldarius. J. Bioenerg. Biomembr. 35:121-31.

23. Hugler, M., R. S. Krieger, M. Jahn, and G. Fuchs. 2003. Characterization of acetyl-CoA/propionyl-CoA carboxylase in Metallosphaera sedula. Carboxylating enzyme in the 3-hydroxypropionate cycle for autotrophic carbon fixation. Eur. J. Biochem. 270:736-44.

24. Kappler, U., L. I. Sly, and A. G. McEwan. 2005. Respiratory gene clusters of Metallosphaera sedula - differential expression and transcriptional organization. Microbiology 151:35-43.

25. Meulenberg, J. J., E. Sellink, N. H. Riegman, and P. W. Postma. 1992. Nucleotide sequence and structure of the Klebsiella pneumoniae pqq operon. Mol. Gen. Genet. 232:284-94.

26. Muir, M. A., T. 1977. Determination of Ferrous Iron in Copper-Process Metallurgical Solutions by the o-Phenanthroline Colorimetric Method Metallurgical transactions. B, Process metallurgy 8B:517-518.

27. Muller, F. H., T. M. Bandeiras, T. Urich, M. Teixeira, C. M. Gomes, and A. Kletzin. 2004. Coupling of the pathway of sulphur oxidation to dioxygen reduction: characterization of a novel membrane-bound thiosulphate:quinone oxidoreductase. Mol. Microbiol. 53:1147-60.

28. Nemati, M., J. Lowenadler, and S. T. Harrison. 2000. Particle size effects in bioleaching of pyrite by acidophilic thermophile Sulfolobus metallicus (BC). Appl. Microbiol. Biotechnol. 53:173-9.

29. Norris, P. R., N. P. Burton, and N. A. Foulis. 2000. Acidophiles in bioreactor mineral processing. Extremophiles 4:71-6.

30. Olson, G. J., J. A. Brierley, and C. L. Brierley. 2003. Bioleaching review part B: progress in bioleaching: applications of microbial processes by the minerals industries. Appl. Microbiol. Biotechnol. 63:249-57.

185

31. Peeples, T. L., and R. M. Kelly. 1995. Bioenergetic Response of the Extreme Thermoacidophile Metallosphaera sedula to Thermal and Nutritional Stresses. Appl. Environ. Microbiol. 61:2314-2321.

32. Purschke, W. G., C. L. Schmidt, A. Petersen, and G. Schafer. 1997. The terminal quinol oxidase of the hyperthermophilic archaeon Acidianus ambivalens exhibits a novel subunit structure and gene organization. J. Bacteriol. 179:1344-53.

33. Rawlings, D. E. 2005. Characteristics and adaptability of iron- and sulfur-oxidizing microorganisms used for the recovery of metals from minerals and their concentrates. Microb. Cell Fact. 4:13.

34. Rawlings, D. E., and D. B. Johnson. 2007. The microbiology of biomining: development and optimization of mineral-oxidizing microbial consortia. Microbiology 153:315-24.

35. Rohwerder, T., T. Gehrke, K. Kinzler, and W. Sand. 2003. Bioleaching review part A: progress in bioleaching: fundamentals and mechanisms of bacterial metal sulfide oxidation. Appl. Microbiol. Biotechnol. 63:239-48.

36. Rouillard, J. M., M. Zuker, and E. Gulari. 2003. OligoArray 2.0: design of oligonucleotide probes for DNA microarrays using a thermodynamic approach. Nucleic Acids Res. 31:3057-62.

37. Schelert, J., V. Dixit, V. Hoang, J. Simbahan, M. Drozda, and P. Blum. 2004. Occurrence and characterization of mercury resistance in the hyperthermophilic archaeon Sulfolobus solfataricus by use of gene disruption. J. Bacteriol. 186:427- 37.

38. Schelert, J., M. Drozda, V. Dixit, A. Dillman, and P. Blum. 2006. Regulation of mercury resistance in the crenarchaeote Sulfolobus solfataricus. J. Bacteriol. 188:7141-50.

186

Figure 5.1: Growth curves for M. sedula cultures in the presence of 10g/L chalcopyrite (CuFeS2) and a headspace containing air, 7% CO2 + balance air, or 7% CO2+14% H2 + balance air (line graphs using y-axis at right). Bars show soluble iron levels in cultures vs. vs abiotic controls as a function of time. Typical standard deviations for cell density measurements is 20% and < 10mg/L for iron assay.

187

Figure 5.2: M. sedula cultures and abiotic controls (ac) in the presence of chalcopyrite. 1 = CO2-supplemented M. sedula culture, 1ac = CO2-supplemented abiotic control, 2 = air-only M. sedula culture, 2ac = air-only abiotic control, 3 = H2 + CO2-supplemented M. sedula culture, 3ac = H2 + CO2-supplemented abiotic control. While soluble iron levels in air-only and CO2 enhanced cultures were not clearly higher than abiotic controls according the iron assay results, visual evidence of Fe3+ precipitation, suggestive of bioleaching, was present.

188

Figure 5.3: Heat plot of electron transfer components involved with oxidation of Fe(II) and RISCs. Heat plots compiled using Array File Maker 4.0 (12). Red indicates levels of high transcription, black = average transcription (set to lsm = 0), and green indicates levels of low transcription, for M. sedula ORFs in the inoculum (d0), at 3 days of growth on CuFeS2 (d3), and at 9 days of growth on CuFeS2 (d9). Lsm = least squares mean of normalized log2 transcription levels.

189

Figure 5.4: Heat plot of ORFs involved in determining M. sedula growth mode on CuFeS2. Heat plots compiled using Array File Maker 4.0 (12). Red indicates levels of high transcription, black = average transcription (set to lsm = 0), and green indicates levels of low transcription, for M. sedula ORFs in the inoculum (d0), at 3 days of growth on CuFeS2 (d3), and at 9 days of growth on CuFeS2 (d9).

190

Chapter 5 Appendix

Bioleaching transcriptomes of Metallosphaera sedula reveal complex mixotrophic physiology

191 Table 5.A1: Blue copper protein (Bcp) ORFs potentially involved with bioleaching. Significant differential transcription is considered to have occurred where log2 fold change has a magnitude greater than 1 (equivalent to a 2-fold change) with significance at or above the Bonferroni (≥5.4 for this data set). A = H2 + CO2 (autotrophic inoculum), CFS3 = H2 + CO2 + CuFeS2 (3 days), CFS9 = H2 + CO2 + CuFeS2 (9 days) Log2 fold change Log2 fold change Log2 fold change (A)-(CFS3) (A) - (CFS9) (CFS3)-(CFS9) M. sedula ORFs [Siga] [Siga] [Siga] Previous upregulation?b putative sulfocyanin Msed _0323 (soxE) -1.3 [4.2] -0.3 [0.4] 1.1 [3.2] none Msed _0826 -0.7 [2.3] -0.3 [0.6] 0.4 [1.1] none putative rusticyanin Msed _0966 -2.8 [4.4] -3.4 [5.6] -0.6 [0.6] none Msed _1206 -0.4 [0.5] 1.1 [2.4] 1.4 [3.5] A-H: 2.1 [9.6], A-M: 2.1 [11.9] a Significance = -log10(p-value). For reference, a significance value of 5.4 correlates to a p-value of 4 x 10-6. b Upregulation reported for H, A, or M conditions represents an average of log2 fold changes and significance values for all 4 combinations of biological repeats (i.e. average of log fold changes for autotrophy vs heterotrophy averages values for individual comparisons A1-H1, A1-H2, A2-H1, and A2-H2)

192 Table 5.A2: ORFs potentially involved with terminal oxidase electron transfer from Fe(II). Significant differential transcription is considered to have occurred where log2 fold change has a magnitude greater than 1 (equivalent to a 2-fold change) with significance at or above the Bonferroni (≥5.4 for this data set). A = H2 + CO2 (autotrophic inoculum), CFS3 = H2 + CO2 + CuFeS2 (3 days), CFS9 = H2 + CO2 + CuFeS2 (9 days) Log2 fold change Log2 fold change Log2 fold change (A)-(CFS3) (A) - (CFS9) (CFS3)-(CFS9) M. sedula ORFs [Siga] [Siga] [Siga] Previous upregulation?b SoxEFGHIM Msed _0319 (soxI) -0.9 [2.8] -0.7 [2.0] 0.1 [0.3] A-H: -1.7 [7.0], H-M: 1.3 [5.7]

Msed _0320 (soxH) -1.0 [2.1] -0.9 [1.7] 0.1 [0.2] none

Msed _0321 (soxG) -0.5 [1.1] -0.2 [0.4] 0.2 [0.5] YFS-YS: 1.6 [10.5], YFS-YKT: 1.2 [7.9], Y- YFS: -1.3 [10.2]

Msed _0322 (soxF) -1.6 [4.0] 1.6 [3.7] 3.2 [8.4] none

Msed _0323 (soxE) -1.3 [4.2] -0.3 [0.4] 1.1 [3.2] none

Msed _0324 (soxM) -1.0 [1.7] 1.9 [4.3] 2.9 [6.9] A-M: 2.1 [6.4], H-M: 2.6 [8.7] FoxAA’BCDEFGHIJ Msed _0467 (mfs transporter) 3.0 [13.8] 2.8 [13.1] -0.2 [0.6] Y-YEF: -1.5 [8.0], A-H: 3.3 [16.5], H-M: -2.5 [13.4]

Msed _0468 (foxH) 0.5 [0.7] 0.8 [1.2] 0.3 [0.3] none

Msed _0469 (foxG) 1.4 [4.7] 1.9 [6.9] 0.5 [1.3] none Msed _0469 2o (foxG) 0.3 [0.9] 0.8 [2.7] 0.4 [1.1] A-H: 1.1 [7.2], A-M: 1.5 [12.9]

Msed _0473 (foxJ) -0.2 [0.7] 0.1 [0.4] 0.3 [0.5] YFS-YS: 1.1 [10.0]

Msed _0474 (foxF) 0.6 [1.1] 1.3 [3.2] 0.7 [1.3] none

Msed _0475 (foxE) 0.4 [0.4] 1.0 [1.8] 0.7 [1.0] none

Msed _0477 (foxD) 2.0 [7.2] 2.6 [9.7] 0.7 [1.9] none

193 (Table 5.A2 continued)

0.3 [1.6] 2.5 [14.9] 2.2 [13.6] YE-YEF: 1.7 [7.3]

Msed _0478 (foxC) 0.9 [4.7] 3.9 [16.8] 3.0 [14.2] YE-YEF: 2.4 [8.1]

Msed _0479 1.2 [3.1] 3.6 [10.6] 2.4 [7.2] YE-YEF: 1.9 [10.5]

Msed _0480 (foxB) 0.1 [0.1] 1.5 [4.3] 1.5 [4.1] Y-YFS: 1.9 [9.7]

Msed _0482 0.6 [1.6] 0.5 [1.0] -0.2 [0.3] none

Msed _0483 (foxI) -1.6 [6.7] -0.9 [3.5] 0.7 [2.2] none

Msed _0484 (foxA) 1.5 [3.2] 2.4 [5.6] 0.8 [1.4] YFS-YS: -3.1 [6.4], YFS-YKT: -2.5 [4.8], Msed _0485 (foxA’) Y-YFS: 3.9 [10.6] -3.2 [6.5] -3.5 [7.3] -0.4 [0.4] none Msed _0486 -2.1 [6.0] -1.3 [3.3] 0.8 [1.7] none Msed _0487 (oxidoreductase) -0.7 [3.7] -0.1 [0.1] 0.7 [3.3] YFS-YS: 2.2 [21.1], YFS-YKT: 1.2 Msed _0489 (DNAP β domain) [13.0], Y-YFS: -1.6 [19.4]

194 (Table 5.A2 continued)

SoxNL-CbsAB Msed _0500 (soxN) -2.1 [8.3] -1.6 [6.2] 0.5 [1.4] YFS-YS: 2.1 [11.6], YFS-YKT: 2.4 [13.3], Y-YFS: -1.8 [12.3], H-M: -2.7 [10.3] Msed _0501 (soxL2) -2.2 [9.0] -1.0 [3.8] 1.2 [4.6] none Msed _0502 (cbsA’) -2.2 [3.4] -1.6 [2.2] 0.6 [0.6] YFS-YS: 1.6 [8.8], YFS-YKT:1.4 [7.1], Y-YFS: -1.2 [7.6] Msed _0502 (2o cbsA’) -3.0 [9.7] -1.8 [5.9] 1.2 [3.4] YFS-YS: 1.1 [9.9], A-M:-2.3 [7.5], H- M:-3.0 [10.8] Msed _0503 (cbsB) -1.3 [4.1] 0.2 [0.3] 1.5 [4.8] YFS-YKT: 1.0 [7.8], H-M: -1.3 [6.3] Msed _0504 (cbsA) -2.2 [11.8] -1.7 [9.4] 0.5 [2.5] YFS-YS: 2.4 [9.7], YFS-YKT: 2.7 [11.3], Y-YFS: -1.6 [7.7], A-H: 2.3 [8.0], H-M: Msed _0505 (H+ pump) 0.3 [1.7] -0.6 [4.1] -0.3 [1.5] -2.7 [11.6]

none a Significance = -log10(p-value). For reference, a significance value of 5.4 correlates to a p-value of 4 x 10-6. b Upregulation = >2-fold change AND significance ≥5.4. Upregulation reported for H, A, or M conditions represents an average of log2 fold changes and significance values for all 4 combinations of biological repeats (i.e. average of log fold changes for autotrophy vs heterotrophy averages values for individual comparisons A1-H1, A1-H2, A2-H1, and A2-H2)

195

Table 5.A3: ORFs with significant differential transcription. Significant differential transcription is considered to have occurred where log2 fold change has a magnitude greater than 1 (equivalent to a 2-fold change) with significance at or above the Bonferroni (≥5.4 for this data set). A = H2 + CO2 (autotrophic inoculum), CFS3 = H2 + CO2 + CuFeS2 (3 days), CFS9 = H2 + CO2 + CuFeS2 (9 days) Log2 fold change Log2 fold change Log2 fold change (A)-(CFS3) (A) - (CFS9) (CFS3)-(CFS9) M. sedula ORFs [Siga] [Siga] [Siga] Previous upregulation?b putative pyruvate synthase (PFOR) Msed_0507 (γ) -1.6 [3.6] -1.8 [4.2] -0.2 [0.3] YE-YEF: -1.8 [5.6]

Msed_0508 (δ) -1.9 [3.0] -2.1 [3.3] -0.1 [0.1] YE-YEF: -1.9 [5.8], H-M: -2.0 [6.3]

Msed_0509 (α) -1.0 [0.9] -1.3 [1.3] -0.3 [0.2] YFS-YS: 1.0 [6.8], YE-YEF: -2.0 [5.5], H-M: -1.5 [5.4]

Msed_0510 (β) -1.9 [8.0] -1.5 [6.3] 0.3 [1.0] YE-YEF: -2.0 [6.3], A-H: 2.2 [6.8], H-M: -2.6 [10.0]

Msed_0511 (Radical SAM domain protein) -4.0 [6.9] -2.4 [3.7] 1.6 [2.2] YFS-YS: 1.0 [8.2], YE-YEF: -1.7 [6.0] 27% ID to PqqE in G. oxydans, Felder et al. 2000 Msed_0512 (Radical SAM domain protein) -4.6 [12.6] -3.6 [10.4] 1.0 [2.3] YFS-YKT: 1.5 [11.8], YFS-YS:1.5 [12.2], 24% ID to PqqE in A. calcoaceticus, YFS-Y: 1.4 [11.1] Goosen et al. 1989 Msed_0513 (β-lactamase domain protein) -4.3 [12.7] -3.4 [10.4] 1.0 [2.5] YFS-YKT: 1.7 [9.3], YFS-YS:1.4 [7.8], YFS-Y: 1.5 [10.1], YE-YEF: -2.0 [6.5]

Msed_0514 (peptidase U32) -4.2 [11.7] -3.5 [10.0] 0.7 [1.4] YFS-YKT: 1.1 [10.5], YFS-YS:1.1 [10.4], YFS-Y: 1.0 [10.8], YE-YEF: -1.9 [6.0] Msed_0515 (Radical SAM domain protein) 32% ID to NTD PqqE in K. pneumoniae -2.6 [8.9] -0.9 [2.7] 1.6 [5.5] YFS-YKT: 1.3 [10.0], YFS-YS:1.8 [13.4], (conserved FeS-binding region) YFS-Y: 1.9 [15.3] , YE-YEF: -2.5 [9.4], Meulenberg et al. 1992 A-M: -1.7 [11.2]

Msed_0516 (hypothetical) -2.7 [9.5] -2.1 [7.4] 0.6 [1.5] YE-YEF: -1.0 [7.3]

Msed_0517 (hypothetical) -1.8 [1.4] -1.6 [1.2] 0.2 [0.1] H-M: -1.9 [7.3]

196 (Table 5.A3 continued)

Msed_0518 (hypothetical) -4.6 [13.1] -4.1 [12.0] 0.5 [0.9] YFS-YKT: 1.7 [11.8], YFS-YS:2.0 ATPase-like NTD; flavoprotein-like [14.1], YFS-Y: 2.1 [17.5], YE-YEF: - CTD 3.5 [11.3], A-M: -2.0 [8.8], H-M: -2.0 [8.8] YFS-YKT: -2.6 [10.5], YFS-YS:-3.8 Msed_1905 (hypothetical protein) 0.6 [2.2] 0.7 [3.1] 0.2 [0.4] [15.1], Y-YS: -3.7 [17.4], Y-YKT: -2.5 [10.0] Msed_0371 (hypothetical protein) -0.9 [1.2] 0.6 [0.6] 1.5 [2.3] YFS-YKT: -2.9 [9.3], Y-YKT: -3.2 [10.4], YKT-YS: 1.8 [6.0]

Msed_0372 (DUF395 YeeE/YedE) 0.1 [0.1] 1.4 [4.8] 1.3 [4.5] YFS-YKT: -3.3 [12.1], Y-YS: -1.7 [6.4], Y-YKT: -3.4 [12.4], YKT-YS: 1.7 [6.1]

Msed_0373 (SirA family YeeD/YedF) -0.3 [0.3] 1.2 [1.6] 1.5 [2.2] YFS-YKT: -3.5 [10.8], Y-YKT: -3.5 40% ID to E.coli TusA sulfurtransferase [10.7], YKT-YS: 2.8 [9.6]

Table 5.A4: ORFs implicated in RISC processing. Significant differential transcription is considered to have occurred where log2 fold change has a magnitude greater than 1 (equivalent to a 2-fold change) with significance at or above the Bonferroni (≥5.4 for this data set). A = H2 + CO2 (autotrophic inoculum), CFS3 = H2 + CO2 + CuFeS2 (3 days), CFS9 = H2 + CO2 + CuFeS2 (9 days) Log2 fold change Log2 fold change Log2 fold change (A)-(CFS3) (A) - (CFS9) (CFS3)-(CFS9) M. sedula ORFs [Siga] [Siga] [Siga] Previous upregulation? putative thiosulfate: quinone oxidoreductase (TQO) Msed_0363 (doxD) -0.5 [1.0] -1.0 [3.0] -0.6 [1.3] none Msed _0364 (doxA) -0.8 [3.4] -1.6 [8.0] -0.9 [3.8] none Msed _0374 (doxD) 1.1 [2.3] 1.8 [4.5] 0.7 [1.3] none putative tetrathionate hydrolase Msed _0804 (tetH) 1.0 [3.2] 1.0 [3.1] [0.0] YFS – YS: -3.0 [12.5]; YFS – YKT: -4.9 [19.7] Y-YS: -3.0 [14.8]; Y-YKT: -5.0 [20.0] a Significance = -log10(p-value). For reference, a significance value of 5.4 correlates to a p-value of 4 x 10-6.

197 Table 5.A5: ORFs potentially involved with terminal oxidase electron transfer from RISCs. Significant differential transcription is considered to have occurred where log2 fold change has a magnitude greater than 1 (equivalent to a 2-fold change) with significance at or above the Bonferroni (≥5.4 for this data set). A = H2 + CO2 (autotrophic inoculum), CFS3 = H2 + CO2 + CuFeS2 (3 days), CFS9 = H2 + CO2 + CuFeS2 (9 days) Log2 fold change Log2 fold change Log2 fold change (A)-(CFS3) (A) - (CFS9) (CFS3)-(CFS9) M. sedula ORFs [Siga] [Siga] [Siga] Previous upregulation?b SoxABCDD’L Msed _0284 -1.9 [6.1] -0.6 [1.2] 1.3 [3.9] YFS-YS: -2.0 [8.3], YFS-YKT: -1.8 [7.0]

Msed _0285 (soxD) -1.3 [9.0] -1.0 [6.5] 0.4 [1.8] YFS-YS: -2.4 [11.2], YFS-YKT: -3.9 [17.9], Y- YKT: -1.7 [7.6]; A-H: 1.7 [5.9], H-M: -1.9 [8.2]

Msed _0286 (soxD’) -1.6 [9.9] -1.5 [9.1] 0.2 [0.4] YFS-YS: -1.5 [7.2], YFS-YKT: -2.3 [12.1], H-M: -1.7 [8.1]

Msed _0288 (soxL) -0.6 [5.2] 0.7 [5.9] 1.3 [11.0] none

Msed _0289 (soxA) -0.4 [13.0] 0.3 [10.9] 0.7 [18.8] YFS-YS: -3.2 [13.8], YFS-YKT: -3.8 [16.4] , Y-YS: -1.7 [8.5], Y-YKT: -2.4 [10.1]

Msed _0290 (soxB) -0.4 [1.3] 0.1 [0.2] 0.5 [1.7] none

Msed _0291 (soxC) -0.7 [1.1] 0.2 [0.2] 0.8 [1.6] none

Msed _0292 (DUF1404) -0.2 [0.2] 0.7 [1.1] 0.9 [1.5] A-H: 1.8 [6.2] DoxBCE Msed _2030 (doxE) -2.8 [8.2] -3.5 [10.2] -0.7 [1.6] none

Msed _2031 (doxC) -1.0 [10.0] -1.3 [11.8] -0.2 [1.6] none

Msed _2032 (doxB) -2.1 [10.6] -2.3 [11.4] -0.2 [0.5] Y-YFS: -1.0 [5.6]

Msed _0570 (doxB2) -0.4 [0.7] -1.9 [5.9] -1.5 [4.5] Y-YS: -1.1 [5.7] a Significance = -log10(p-value). For reference, a significance value of 5.4 correlates to a p-value of 4 x 10-6. b Upregulation =>2-fold change AND significance ≥5.4. Upregulation reported for H, A, or M conditions represents an average of log2 fold changes and significance values for all 4 combinations of biological repeats (i.e. average of log fold changes for autotrophy vs heterotrophy averages values for individual comparisons A1-H1, A1-H2, A2-H1, and A2- H2)

198

Table 5.A6: ORFs potentially involved with electron transfer from RISCs. Significant differential transcription is considered to have occurred where log2 fold change has a magnitude greater than 1 (equivalent to a 2-fold change) with significance at or above the Bonferroni (≥5.4 for this data set). A = H2 + CO2 (autotrophic inoculum), CFS3 = H2 + CO2 + CuFeS2 (3 days), CFS9 = H2 + CO2 + CuFeS2 (9 days) Log2 fold change Log2 fold change Log2 fold change (A)-(CFS3) (A) - (CFS9) (CFS3)-(CFS9) M. sedula ORF [Siga] [Siga] [Siga] Previous upregulation?b putative sre-/dms-like cluster Msed _0810 (regulator) -1.6 [3.4] 0.2 [0.2] 1.9 [4.0] YFS-YKT: -2.1 [7.8]

Msed _0811 (GTP-binding) -0.6 [0.8] -0.6 [0.8] 0.0 [0.0] none

Msed _0812 (memb. anchor) -2.9 [3.9] 1.4 [1.5] 4.4 [6.3] YFS-YS: -2.0 [6.3], YFS-YKT: -4.2 [14.7] , Y-YS: -1.6 [5.8], Y-YKT: -3.8 [13.3]

Msed _0813 (TPR domain) -1.2 [4.4] -1.3 [4.8] -0.1 [0.1] none

Msed _0814 (dmsA-like) -2.2 [4.5] 0.7 [1.0] 2.9 [6.3] YFS-YS: -2.2 [8.4], YFS-YKT: -2.9 [11.4], Y-YS: -2.4 [11.1], Y-YKT: -3.0 [12.1]

Msed _0815 (sreB/dmsB-like) -2.0 [5.4] 0.8 [1.7] 2.8 [7.9] YFS-YS: -2.0 [9.5], YFS-YKT: -3.2 [15.8], Y-YS: -2.1 [12.3], Y-YKT: -3.3 [16.3]

Msed _0816 (sreE/dmsD-like) -1.5 [2.9] 0.1 [0.1] 1.6 [3.2] YFS-YS: -1.3 [8.9], YFS-YKT: -1.7 [12.4], Y-YS: -1.3 [9.3], Y-YKT: -1.8 [12.8]

Msed _0817 (sreD-like) -1.4 [6.2] -0.5 [1.8] 0.9 [3.4] YFS-YKT: -1.3 [7.5], Y-YKT: -1.4 [8.4]

Msed _0818 0.3 [0.4] 0.4 [0.4] 0.0 [0.0] none

199 (Table 5.A6 continued)

heterodisulfide-like complex Msed _1542 (hdrB1-like) -1.9 [4.5] 0.6 [1.0] 2.5 [6.3] YFS-YS: -2.1 [5.5], YFS-YKT: -2.5 [7.0] , Y-YS: -2.0 [6.8], Y-YKT: -2.4 [6.8]

Msed _1543 (hdrC1-like) -0.9 [2.4] 1.1 [3.1] 1.9 [6.6] YFS-YS: -3.5 [10.7], YFS-YKT: -3.7 [11.1], Y-YS: -2.5 [9.0], Y-YKT: -2.7 [7.6]; A-M:1.8 [6.1]

Msed _1544 -1.2 [1.9] 1.2 [2.0] 2.4 [4.9] YFS-YS: -2.2 [6.0], YFS-YKT: -2.5 [6.9] , Y-YS: -1.7 [5.5]

Msed _1545 (hdrA-like) -0.6 [1.7] 0.7 [2.3] 1.3 [5.0] none

Msed _1546 (hdrB2-like) -0.9 [4.5] 0.8 [3.8] 1.7 [8.9] YFS-YKT: -2.3 [5.9]

Msed _1547 (hdrC2-like) -1.2 [3.9] -0.4 [0.8] 0.8 [2.2] none

Msed _1548 (regulator?) -0.9 [21.6] -0.8 [21.3] 0.1 [3.0] YFS-YS: -3.0 [10.9], YFS-YKT: -3.3 [12.1]

Msed _1549 -0.7 [18.8] -0.6 [17.2] 0.1 [3.8] YFS-YS: -2.8 [6.7], YFS-YKT: -4.2 [10.9], Y-YKT: -2.5 [5.8]

Msed _1550 0.1 [0.0] 1.1 [0.5] 1.0 [0.4] YFS-YS: -2.3 [5.6], YFS-YKT: -3.6 [10.0] a Significance = -log10(p-value). For reference, a significance value of 5.4 correlates to a p-value of 4 x 10-6. b Upregulation reported for H, A, or M conditions represents an average of log2 fold changes and significance values for all 4 combinations of biological repeats (i.e. average of log fold changes for autotrophy vs heterotrophy averages values for individual comparisons A1-H1, A1-H2, A2-H1, and A2-H2)

200

Table 5.A7: Potential ORFs involved with CO2 fixation via the 3-hydroxypropionate/4-hydroxybutyrate cycle. Significant differential transcription is considered to have occurred where log2 fold change has a magnitude greater than 1 (equivalent to a 2-fold change) with significance at or above the Bonferroni (≥5.4 for this data set). A = H2 + CO2 (autotrophic inoculum), CFS3 = H2 + CO2 + CuFeS2 (3 days), CFS9 = H2 + CO2 + CuFeS2 (9 days). Log2 fold change Log2 fold change Log2 fold change (A)-(CFS3) (A) - (CFS9) (CFS3)-(CFS9) M. sedula ORFs [Siga] [Siga] [Siga] Previous upregulation?b acetyl/propionyl-CoA carboxylase Msed _0147 0.0 [0.1] 2.5 [13.0] 2.5 [13.2] A-H: 3.1 [15.9], A-M: 3.5 [20.9] Msed _0148 0.6 [1.5] 2.4 [8.0] 1.7 [5.7] A-H: 2.8 [15.6], A-M: 3.5 [22.0] Msed _1375 -1.0 [4.9] 1.3 [6.9] 2.2 [11.6] none malonyl-/succinyl-CoA reductase Msed _0709 -1.0 [6.9] 1.5 [10.0] 2.5 [14.8] A-H: 4.6 [17.7], A-M: 3.4 [14.0] 3-hydroxypropionate synthetase Msed _1456 -0.2 [0.2] 1.8 [3.3] 2.1 [3.9] A-H: 2.7 [9.4], A-M: 1.8 [ 6.2] 3-hydroxypropionyl-CoA dehydratase Msed_2001 0.2 [0.3] 0.3 [0.7] 0.2 [0.3] Y-YFS: 1.4 [5.4] acryloyl-CoA reductase Msed_1426 0.6 [0.9] 1.0 [1.6] 0.3 [0.4] none methylmalonyl-CoA epimerase Msed _0639 0.8 [1.9] 0.8 [1.8] 0.0 [0.1] none methylmalonyl-CoA mutase Msed _0638 0.5 [0.7] 0.9 [1.5] 0.4 [0.4] Msed _2055 / Msed_2056 -0.5 [0.5] / 0.1 [0.1] 0.2 [0.2] / 0.4 [1.3] 0.7 [0.9] / 0.3 [1.0] none succinate semialdehyde reductase Msed _1424 -1.0 [1.7] 1.2 [2.3] 2.1 [5.0] none 4-hydroxybutyryl-CoA synthetase Msed _1422 0.1 [0.1] 2.4 [5.8] 2.4 [5.7] A-H: 3.0 [11.0], A-M: 2.6 [10.6] 4-hydroxybutyryl-CoA dehydratase Msed _1321 -0.6 [0.5] 1.3 [1.4] 1.9 [2.3] A-H: 2.9 [9.5], A-M: 3.2 [12.3] Crotonyl-hydratase (3 candidates) Msed_0336 0.3 [0.5] 0.3 [0.5] 0.0 [0.0] none Msed _0399 1.7 [3.4] 0.7 [1.1] -1.0 [1.6] YKS-YS:-1.3 [6.3], YKT-YS:-1.2 Msed _0566 0.4 [0.9] 0.2 [0.4] -0.2 [0.4] [7.1] none

201 (Table 5.A7 continued)

3-hydroxybutyryl-CoA dehydrogenase Msed_1993 / Msed_1994 1.5 [3.3] / 0.9 [2.1] 2.5 [6.5] / 4.0 [11.2] 1.1 [2.1] / 3.1 [9.2] A-H: 2.0 [6.5] / A-H: 1.9[5.6] acetoacetyl-CoA β-ketothiolase Msed _0656 -1.0 [1.3] 0.9 [1.1] 1.8 [3.2] none Msed_2087 1.1 [2.1] 2.7 [7.0] 1.7 [3.9] A-H: 2.9 [9.8], A-M: 3.0 [11.6] a Significance = -log10(p-value). For reference, a significance value of 5.4 correlates to a p-value of 4 x 10-6. b Upregulation reported for H, A, or M conditions represents an average of log2 fold changes and significance values for all 4 combinations of biological repeats (i.e. average of log fold changes for autotrophy vs heterotrophy averages values for individual comparisons A1-H1, A1-H2, A2-H1, and A2-H2)

Table 5.A8: Carbonic anhydrases annotated in the M. sedula genome. Significant differential transcription is considered to have occurred where log2 fold change has a magnitude greater than 1 (equivalent to a 2-fold change) with significance at or above the Bonferroni (≥5.4 for this data set). A = H2 + CO2 (autotrophic inoculum), CFS3 = H2 + CO2 + CuFeS2 (3 days), CFS9 = H2 + CO2 + CuFeS2 (9 days) Log2 fold change Log2 fold change Log2 fold change (A)-(CFS3) (A) - (CFS9) (CFS3)-(CFS9) M. sedula ORFs [Siga] [Siga] [Siga] Previous upregulation? β class carbonic anhydrase msed_0390 -0.6 [1.6] 1.2 [4.6] 1.8 [7.1] none γ class carbonic anhydrase 0.7 [2.7] 0.4 [1.2] -0.3 [0.9] none msed_1618 a Significance = -log10(p-value). For reference, a significance value of 5.4 correlates to a p-value of 4 x 10-6.

202

Table 5.A9: ORFs up-regulated on heterotrophy in M. sedula genome. Significant differential transcription is considered to have occurred where log2 fold change has a magnitude greater than 1 (equivalent to a 2-fold change) with significance at or above the Bonferroni (≥5.4 for this data set). A = H2 + CO2 (autotrophic inoculum), CFS3 = H2 + CO2 + CuFeS2 (3 days), CFS9 = H2 + CO2 + CuFeS2 (9 days). Log2 fold Log2 fold Log2 fold change change change (A)-(CFS3) (A) - (CFS9) (CFS3)-(CFS9) M. sedula ORFs [Siga] [Siga] [Siga] Previous upregulation?b

Msed_0268: Hypothetical protein -1.4 [6.4] -1.1 [4.7] 0.3 [1.0] H-M: 2.3 [6.5]

Msed_0270: acetyl-CoA acetyltransferase -1.3 [3.3] -1.6 [4.2] -0.3 [0.4] none non-specific lipid/sterol carrier protein

Msed_0319: Hypothetical protein -0.9 [2.8] -0.7 [2.0] 0.2 [0.3] A-H: -1.7 [7.0], H-M: 1.3 [5.7] SoxI Msed_0322: Rieske (2Fe-2S) domain protein -1.6 [4.0] 1.6 [3.7] 3.2 [8.4] Y-YKS: -1.7 [6.5], YFS-YKS:-1.5 [6.4], SoxF YKS-YKT: 1.8 [8.2], YKS-YS: 2.1 [8.3]

Msed_0333: FAD dependent oxidoreductase -0.3 [1.4] -0.1 [0.3] 0.2 [0.8] A-H: -2.1 [6.9] Flavoprotein dehydrogenase-like protein

Msed_0349: Hypothetical protein -0.9 [4.9] -0.3 [0.9] 0.7 [3.1] H-M: -1.9 [7.3]

Msed_0850: Hypothetical protein -1.1 [2.5] -1.5 [3.9] -0.4 [0.7] A-H: -2.3 [9.2], A-M: -1.6 [6.2]

Msed_0855: methyltransferase type 11 -0.7 [3.2] -0.9 [4.8] -0.3 [0.8] A-H: -2.1 [7.8], H-M: 1.5 [5.3] NAD(P)-binding Msed_0858: Hypothetical protein -0.7 [1.3] -0.6 [1.1] 0.1 [0.1] none

Msed_0900: Hypothetical protein -1.4 [4.9] -2.6 [9.5] -1.2 [4.2] A-H: -1.5 [7.5], H-M: 1.2 [6.9], YKS-YS:-1.3 [12.9], YKT-YS: -1.2 [14.4]

203 (Table 5.A9 continued)

Msed_0993: Major facilitator transporter -2.0 [5.2] 0.0 [0.0] 2.0 [5.2] H-M: 1.4 [5.7] similarities to organic substrate transporters

Msed_1037: acetolactate synthase, large subunit -0.4 [1.8] -0.1 [0.2] 0.4 [1.4] A-H: -2.6 [6.0]

Msed_1095: major facilitator transporter -1.2 [3.0] -1.3 [3.4] -0.1 [0.2] A-H: -1.9 [7.7]

Msed_1177: FAD dependent oxidoreductase -0.3 [0.6] -1.0 [3.0] -0.7 [1.9] A-H: -1.6 [8.1] 41% identity to glycerol-3-phosphate dhase in E. coli Msed_1178: glycerophosphodiester -0.5 [4.1] -0.6 [5.3] -0.1 [0.5] A-H: -1.4 [8.1], A-M: -1.2 [7.5], YFS-YS: phosphodiesterase 1.1 [11.4], YKS-YS: 1.0 [10.1] potentially converts phospholipids to G-3-P

Msed_1180: ABC transporter related -0.5 [0.8] -0.5 [0.9] -0.1 [0.1] A-H: -2.3 [7.6] similar to ATPase for bacterial G-3-P import system

Msed_1227: TM helix repeat-containing protein -1.1 [5.2] -1.3 [6.3] -0.2 [0.6] A-H: -2.4 [7.4] membrane protein with 6 predicted TMs

Msed_1254: Hypothetical protein -1.7 [4.0] -1.6 [3.8] 0.1 [0.1] A-H: -1.6 [6.0]

Msed_1581: succinyl-CoA synthetase (ADP- -1.2 [1.8] -1.6 [2.9] -0.4 [0.5] none forming) α subunit Msed_1582: succinyl-CoA synthetase (ADP- -1.3 [2.4] -2.0 [4.4] -0.7 [1.1] A-H: -2.3 [5.5], YKT-YS: -1.3 [5.4] forming) β subunit

Msed_1733: transcription initiation factor IIB -0.5 [2.7] -0.3 [1.5] 0.2 [0.7] A-H: -2.0 [6.3]

204 (Table 5.A9 continued)

Msed_1772: major facilitator transporter -1.9 [4.6] -2.9 [7.4] -1.0 [1.9] none

Msed_1775: 3,4-dihydroxyphenylacetate 2,3- -3.5 [7.5] -4.0 [8.5] -0.5 [0.5] A-H: -2.2 [9.3], H-M: 1.8 [8.1] dioxygenase

Msed_1822: hypothetical protein -0.3 [0.4] -0.1 [0.1] 0.2 [0.3] A-H: -1.6 [5.7]

Msed_1833: hypothetical protein -0.8 [2.5] -0.3 [0.7] 0.4 [1.2] A-H: -1.9 [6.2] Sulfolobus transposase-like domain

Msed_1854: hypothetical protein -0.3 [1.4] -0.3 [1.4] 0.0 [0.0] A-H: -2.0 [7.6] similar to bacterial glycosyl hydrolase (family 1)

Msed_2074: Glu/Leu/Phe/Val dehydrogenase, C 0.1 [0.1] -2.8 [5.1] -3.0 [5.3] A-H: -2.1 [5.7], YKT-YS: -2.0 [6.0] terminal; similar to Glu dhase in S. solfataricus, Maras et al. 1992

Msed_2282: hypothetical protein -1.0 [4.0] -1.0 [4.4] -0.1 [0.1] A-H: -1.5 [5.6] similar to phage-integrase from SSV1 a Significance = -log10(p-value). For reference, a significance value of 5.4 correlates to a p-value of 4 x 10-6. b Upregulation reported for H, A, or M conditions represents an average of log2 fold changes and significance values for all 4 combinations of biological repeats (i.e. average of log fold changes for autotrophy vs heterotrophy averages values for individual comparisons A1-H1, A1-H2, A2-H1, and A2-H2)

205

Table 5.A10: Putative biosynthesis ORFs upregulated on autotrophy in M. sedula genome. Significant differential transcription is considered to have occurred where log2 fold change has a magnitude greater than 1 (equivalent to a 2-fold change) with significance at or above the Bonferroni (≥5.4 for this data set). A = H2 + CO2 (autotrophic inoculum), CFS3 = H2 + CO2 + CuFeS2 (3 days), CFS9 = H2 + CO2 + CuFeS2 (9 days) Log2 fold Log2 fold change change Log2 fold change (A)-(CFS3) (A) - (CFS9) (CFS3)-(CFS9) M. sedula ORFs [Siga] [Siga] [Siga] Previous upregulation?b Nitrogen assimilating amino acids Msed_1612: L-glutamine synthetase 2.3 [8.0] 2.9 [9.8] 0.6 [1.3] A-H: 2.3 [7.7], A-M: 2.1 [7.4] glnA; similar to S. solfataricus, Sanangelantoni et al. 1990 Msed_1884: glutamate synthase (NADPH) GltB2 subunit -0.2 [0.2] 2.2 [5.9] 2.4 [6.5] A-H: 2.3 [6.0] A-M: 2.3 [6.8]

Msed_1885: glutamate synthase (NADPH) GltB1 subunit -0.1 [0.2] 0.9 [2.4] 1.0 [2.9] A-M: 1.8 [9.4]

Sulfur assimilating amino acids Msed_0960: uroporphyrin-III /tetrapyrrole methyltransferase 1.8 [8.1] 1.9 [8.5] 0.1 [0.2] A-H: 1.9 [5.5], H-M: -2.0 [7.1], cysG-like; Auernik et al. 2008 YE-YEF:-1.3 [9.3]

Msed_0961: sulfite reductase (NADPH) beta subunit 2.7 [13.0] 3.0 [14.1] 0.3 [1.1] A-H: 4.6 [18.4], H-M: -3.4 [14.6], cysI-like; Auernik et al. 2008 YE-YEF:-1.6 [8.5]

Msed_0962: phosphoadenosine phosphosulfate reductase 2.0 [5.0] 2.6 [6.7] 0.6 [0.9] A-H: 3.4 [13.9], H-M: -2.5 [11.2], cysH-like; Auernik et al. 2008 YE-YEF:-1.7 [5.9]

Msed_0963: sat sulfate adenylyltransferase 2.7 [11.1] 3.0 [12.1] 0.3 [0.8] A-H: 3.2 [10.1], H-M: -1.8 [5.4] cysN-like; Auernik et al. 2008

Msed_0672: homoserine kinase also used in threonine 0.9 [1.2] 2.4 [4.9] 1.5 [2.8] A-H: 3.3 [11.7], A-M: 3.4 [14.2], Y- biosynthesis YKT: -1.8 [6.8], YFS-YKT: -1.9 [7.4]

Msed_0673: hypothetical protein cystathionine gamma synthase 1.1 [1.7] 2.0 [4.2] 1.0 [1.6] A-H: 2.7 [7.6], A-M: 2.4 [7.4]

Msed_2248: methionine synthase 2.7 [3.8] 2.6 [3.5] -0.1 [0.1] none

206 (Table 5.A10 continued)

Msed_2249: 5-methyltetrahydropteroyltriglutamate-- 2.2 [6.5] 2.1 [6.1] -0.1 [0.2] A-M: 2.2 [7.3], YKS-YS:-1.2[5.6], homocysteine methyltransferase YKT-YS: -1.2 [6.1]

Msed_2250: DNA polymerase sliding clamp subunit A 1.4 [2.9] 1.0 [1.8] -0.4 [0.5] A-M: 2.1 [7.0], YFS-YS:-1.3[8.8], YKS-YS:-1.5[10.5], YKT-YS: -1.3 [10.9]

Aliphatic amino acids Msed_0623: trans-homoaconitate synthase 0.9 [2.0] 0.7 [1.4] -0.2 [0.2] A-M: 2.1 [7.1] LeuA-/CimA-like Msed_1765: 2-isopropylmalate synthase 1.4 [1.9] 2.1 [3.4] 0.7 [0.7] A-H: 2.9 [11.2], H-M: -2.2 [9.1] LeuA-CimA-like

Msed_0828: aminotransferase, class V 1.4 [1.4] 0.8 [0.7] -0.6 [0.5] none

Msed_0829: 3-isopropylmalate dehydratase small subunit 0.9 [2.0] 1.8 [5.5] 1.0 [2.4] H-M: -1.2 [5.4] LeuD-like; similar to G. sulfurreducens, Risso et al. 2008 Msed_0830: 3-isopropylmalate dehydratase large subunit 0.6 [1.5] 1.7 [6.0] 1.1 [3.5] none LeuC-like; similar to G. sulfurreducens, Risso et al. 2008

Msed_1927: ketol-acid reductoisomerase 0.2 [0.4] 1.1 [4.4] 0.9 [3.4] none IlvC-like; similar to G. sulfurreducens, Risso et al. 2008 Msed_1928: hypothetical protein 0.3 [0.3] 1.7 [3.1] 1.4 [2.3] A-H: 2.0 [5.5], A-M: 2.1 [7.1] IlvH-like; similar to G. sulfurreducens, Risso et al. 2008 Msed_1929: acetolactate synthase catalytic subunit -0.1 [0.1] 1.6 [3.7] 1.7 [3.9] none IlvI-like; similar to G. sulfurreducens, Risso et al. 2008

Aromatic amino acids Msed_1688: phosphoribosylanthranilate isomerase 1.0 [1.8] 0.9 [1.5] -0.1 [0.1] none TrpF-like Msed_1689: anthranilate phosphoribosyltransferase 1.5 [3.1] 1.2 [2.4] -0.3 [0.3] none TrpD-like

207 (Table 5.A10 continued)

Hydrophilic (+) R group amino acids Msed_0165: acetylglutamate kinase 1.9 [4.5] 2.3 [5.5] 0.3 [0.4] A-H: 2.0 [6.5], A-M: 2.1 [8.6] involved in Lys and Arg biosynthesis Msed_0168: L-2-aminoadipate N-acetyltransferase -0.2 [0.3] 1.4 [3.7] 1.6 [4.3] A-M: 2.2 [6.2] LysX-like Msed_0169: N2-acetyl-L-Lys aminotransferase/ 0.8 [1.1] 1.3 [2.2] 0.5 [0.6] A-M: 2.1 [6.4] acetylornithine; ArgD-like, involved in Lys and Arg biosynthesis

Msed_1988: argininosuccinate lyase 0.4 [0.8] 1.3 [4.1] 0.9 [2.6] none ArgH-like Riboflavin biosynthesis Msed_0046: 3,4-dihydroxy-2-butanone 4-phosphate 2.1 [6.0] 2.1 [5.9] 0.0 [0.0] A-H: 2.1 [7.7], A-M: 3.1 [14.7] synthase RibB-like 1.6 [4.3] 1.6 [4.2] 0.0 [0.0] A-H: 1.8 [5.6], A-M: 2.1 [8.3] Msed_0047: riboflavin synthase RibC-like 2.3 [5.4] 2.2 [5.1] -0.1 [0.1] A-M: 2.6 [9.3] Msed_0048: riboflavin synthase subunit beta RibH Thiamine biosynthesis Msed_2221: ribulose-1,5-biphosphate synthetase 0.5 [1.3] 1.0 [3.1] 0.5 [1.1] A-H: 2.6 [11.3], A-M: 3.7 [20.1] putative thiazole biosynthetic enzyme Msed_0906: thiamine biosynthesis protein ThiC -0.4 [0.4] 0.1 [0.1] 0.6 [0.5] A-H: 2.8 [13.7], A-M: 3.0 [17.5] a Significance = -log10(p-value). For reference, a significance value of 5.4 correlates to a p-value of 4 x 10-6. b Upregulation reported for H, A, or M conditions represents an average of log2 fold changes and significance values for all 4 combinations of biological repeats (i.e. average of log fold changes for autotrophy vs heterotrophy averages values for individual comparisons A1-H1, A1-H2, A2-H1, and A2-H2)

208

Table 5.A11: ORFs up-regulated or down-regulated on mixotrophy in M. sedula genome. Significant differential transcription is considered to have occurred where log2 fold change has a magnitude greater than 1 (equivalent to a 2-fold change) with significance at or above the Bonferroni (≥5.4 for this data set). A = H2 + CO2 (autotrophic inoculum), CFS3 = H2 + CO2 + CuFeS2 (3 days), CFS9 = H2 + CO2 + CuFeS2 (9 days) Log2 fold change Log2 fold change Log2 fold change (A)-(CFS3) (A) - (CFS9) (CFS3)-(CFS9) M. sedula ORFs [Siga] [Siga] [Siga] Previous upregulation?b

Msed_0056: DKCLD domain containing protein 1.8 [3.1] 1.0 [1.3] -0.8 [1.0] none pseudouridine synthase (α subunit) Msed_0057: PUA domain containing protein 0.9 [2.2] -0.1 [0.2] -1.0 [2.8] A-M: -1.9 [7.9] pseudouridine synthase (β subunit) Msed_0058: methyltransferase small 0.5 [2.5] 0.1 [0.1] -0.4 [2.1] A-M: -1.4 [6.3], H-M: -1.4 [6.1] rRNA methyltransferase A-M: -2.8 [13.3], H-M: -2.0 [8.4], Msed_0428: NADPH-dependent FMN reductase -0.3 [0.5] -0.3 [0.6] 0.0 [0.0] Y-YKS: -1.4 [8.0], YFS-YKS:-1.4 [8.3], WrbA-like YKS-YS:1.6 [9.5], YKS-YKT: 1.0 [5.6]

Msed_0500: cytochrome b/b6 domain protein -2.1 [8.3] -1.6 [6.2] 0.5 [1.4] H-M: -2.7 [10.3], SoxN cytochrome b Y-YFS: -1.8 [12.3], YFS-YKS:2.3 [15.2], YFS-YKT: 2.4 [13.3],YFS-YS: 2.1 [11.6]

Msed_0502: pseudogene -3.0 [9.7] -1.8 [5.9] 1.2 [3.4] A-M: -2.3 [7.5], H-M: -3.0 [10.8], Cbs A-like cytochrome b YFS-YS: 1.1[9.9]

Msed_0517: Hypothetical protein -1.8 [1.4] -1.6 [1.2] 0.2 [0.1] H-M: -1.9 [7.3] NTP-binding NTD

Msed_0849: hypothetical protein -1.0 [2.4] -0.9 [2.1] 0.1 [0.1] H-M: -1.3 [5.7], Y-YFS:-1.5 [10.3], ABC transporter, permease subunit (cobalt?) Y-YKS: -1.1 [5.8], YFS-YS: 2.1 [12.1], YKS-YS:1.7 [9.9] , YKT-YS: 1.1 [7.4]

Msed_0997: Rieske [2Fe-2S] protein -0.3 [1.7] -0.3 [2.0] 0.0 [0.1] A-M: -2.7 [14.2] sulredoxin (82% ID to S. tokodaii, Iwasaki et al. 1996)

209 (Table 5.A11 continued)

Msed_1205: hypothetical protein -0.8 [3.7] -0.5 [2.2] 0.3 [0.9] A-M: -1.3 [5.8] (unique 28-Feb-2009)

Msed_1364: GMP synthase, α subunit 0.5 [1.9] 0.2 [0.6] -0.3 [0.8] A-M: -1.8 [5.8]

Msed_1365: dual specificity protein phosphatase -0.2 [0.3] -0.7 [1.3] -0.5 [0.8] none possibly involved with signal transduction?

Msed_1366: Endonuclease V 0.0 [0.0] -0.4 [0.3] -0.4 [0.4] none

Msed_2054: hypothetical protein -1.1 [1.6] -1.6 [2.9] -0.6 [0.7] H-M: -1.3 [6.8], Y-YKT: -1.3 [8.3], YFS-YKT:-1.3 [7.9], YKS-YKT:-1.0 [7.5], YKT-YS: 1.1[8.2]

Msed_2232: CopG/Arc/MetJ family transcriptional 0.3 [0.6] 1.0 [2.8] 0.6 [1.5] A-M: -1.6 [6.0] regulator

1.4 [2.5] 1.1 [1.7] -0.3 [0.4] A-M: 2.5 [11.1], H-M: 1.5 [5.4], Msed_0077 (2p): exosome complex exonuclease Rrp41 YKS-YS:-1.6 [5.5],YKT-YS: -1.6 [6.6]

Msed_0227: orotidine 5'-phosphate decarboxylase 1.9 [5.6] 0.8 [1.8] -1.1 [2.7] A-M: 2.0 [8.2], H-M: 1.7 [6.4] 3-hexulose 6-phosphate synthase (HPS)

-1.0 [1.7] 1.9 [4.3] 2.9 [6.9] A-M: 2.1 [6.4], H-M: 2.6 [8.7], Msed_0324: cytochrome c oxidase YFS-YKS: -2.1[9.7], YKS-YKT: SoxM cytochrome oxidase 1.9[8.5]

Msed_0328: hypothetical protein -0.8 [1.1] 3.0 [6.4] 3.8 [8.2] A-M: 2.1 [9.8], H-M: 1.6 [6.9]

Msed_0743: FAD-dependent pyridine nucleotide- 0.6 [0.2] 0.7 [0.3] 0.1 [0.0] A-M: 3.1 [12.1] disulphide oxidoreductase; thioredoxin reductase?

210 (Table 5.A11 continued)

Msed_1201: pyruvate ferredoxin/flavodoxin -1.2 [4.0] 0.4 [0.8] 1.5 [5.7] A-M: 1.7 [7.2], H-M: 1.8 [8.0] oxidoreductase, δ subunit ; PorD-like at CTD only

Msed_1491: metallophosphoesterase 0.6 [1.1] 0.7 [1.4] 0.1 [0.1] A-M: 2.5 [10.9] possibly involved with signal transduction?

Msed_1659: SpoVT/AbrB domain protein -0.9 [1.5] -0.6 [0.8] 0.3 [0.4] A-M: 2.2 [9.4], H-M: 1.9 [7.5], regulatory protein? Y-YFS: 1.1 [6.3], YFS-YS:-1.5 [9.0], YFS-YKT:-1.1 [6.0]

Msed_2221: ribulose-1,5-biphosphate synthetase 0.5 [1.3] 1.0 [3.1] 0.5 [1.1] A-H: 2.6 [11.3], A-M: 3.7 [20.1] thiazole biosynthesis protein a Significance = -log10(p-value). For reference, a significance value of 5.4 correlates to a p-value of 4 x 10-6. b Upregulation reported for H, A, or M conditions represents an average of log2 fold changes and significance values for all 4 combinations of biological repeats (i.e. average of log fold changes for autotrophy vs heterotrophy averages values for individual comparisons A1-H1, A1-H2, A2-H1, and A2-H2)

211

Table 5.A12: Potential ORFs involved with NiFe hydrogenases in M. sedula. Significant differential transcription is considered to have occurred where log2 fold change has a magnitude greater than 1 (equivalent to a 2-fold change) with significance at or above the Bonferroni (≥5.4 for this data set). A = H2 + CO2 (autotrophic inoculum), CFS3 = H2 + CO2 + CuFeS2 (3 days), CFS9 = H2 + CO2 + CuFeS2 (9 days) Log2 fold change Log2 fold change Log2 fold change (A)-(CFS3) (A) - (CFS9) (CFS3)-(CFS9) M. sedula ORFs [Siga] [Siga] [Siga] Previous upregulation?b putative membrane-bound hydrogenase msed_0944 (β) -0.9 [0.4] 1.4 [0.7] 2.3 [1.3] A-H: 3.7 [12.5], H-M: -2.7 [9.9], Y-YS: - 1.4 [10.5]; YFS-YS: -1.1 [7.7], YKT-YS: -1.6 [12.2], YKS-YS: -1.4 [11.0]

msed_0945 (α) -0.6 [8.6] 1.6 [17.0] 2.2 [20.4] A-H: 2.9 [14.6], H-M: -2.0 [10.5]

msed_0946 (anchor) -0.7 [12.9] 1.1 [16.9] 1.9 [22.1] A-H: 3.4 [10.1], A-M: 2.1 [5.8], Y-YS: -1.3 [9.0], YKS-YS: -1.2 [6.9] Fe-S oxidoreductase-like protein msed_0947 -0.8 [0.6] 1.5 [1.3] 2.3 [2.4] A-H: 2.0 [9.1], YFS-YS: -1.1 [7.7], YKS-YS: -1.4 [11.0] associated hypothetical proteins msed_0948 -0.9 [8.4] 1.2 [10.0] 2.1 [15.6] A-H: 3.6 [14.5], H-M: -2.2 [8.3] msed_0949 -0.8 [7.5] 1.2 [10.6] 2.0 [15.5] A-H: 4.1 [18.7], A-M: 3.0 [14.7] msed_0950 -1.4 [3.7] 1.2 [2.9] 2.5 [7.5] A-H: 1.8 [5.9] putative soluble hydrogenase msed_0923 (α) -1.0 [2.6] -0.9 [2.5] 0.0 [0.0] none msed_0924 (β) -1.6 [3.0] -1.7 [3.2] -0.1 [0.1] none associated hypothetical proteins msed_0921 -0.8 [1.4] -0.8 [1.4] 0.0 [0.0] none msed_0922 -0.6 [1.2] -0.8 [1.8] -0.2 [0.3] none

212 (Table 5.A12 continued)

putative accessory proteins msed_0913 (hypA) 0.8 [2.3] 0.6 [1.7] -0.2 [0.3] none msed_0915 (hypF) -0.5 [0.4] -0.7 [0.7] -0.2 [0.2] Y-YFS: -1.6 [8.8], YFS-YKS:1.1 [5.6], YFS-YS: 2.1 [9.7], YFS-YKT:1.5 [6.1]

msed_0916 (maturation protease) -0.9 [2.6] -1.1 [3.0] -0.1 [0.2] none msed_0917 (hypC) -0.9 [4.1] -0.8 [3.6] 0.1 [0.2] none msed_0918 (hypC2) -1.5 [4.2] -1.4 [3.8] 0.1 [0.2] H-M: -2.5 [7.4] msed_0930 (hypD) -0.8 [1.9] -0.3 [0.5] 0.5 [1.0] A-H: 2.5 [5.7] msed_0931 (hypC3) -0.1 [0.0] 0.0 [0.0] 0.0 [0.0] A-H: 3.0 [8.0] msed_0933 (hypE) -1.2 [2.7] 0.0 [0.0] 1.2 [2.7] A-H: 2.0 [5.7], A-M: 1.8 [5.7] Other related proteins msed_0914 1.4 [4.4] 0.6 [1.5] -0.8 [2.0] none (similarities to cytosolic Fe-S cluster assembly proteins) msed_0919 -1.1 [1.6] -0.7 [0.9] 0.4 [0.4] none (pqqE-like protein with CX3CX2C and CX2CX3C FeS cluster-binding motifs)

213 (Table 5.A12 continued)

Other gene neighborhood proteins Msed_0920 (pseudo-NiFe-α ) -1.5 [3.8] 0.2 [0.3] 1.7 [4.5] none Msed_0925 (hypothetical) -1.7 [2.8] -1.8 [3.1] -0.1 [0.1] none Msed_0926 (HTH regulator) -0.8 [10.6] -0.9 [10.6] 0.0 [0.1] H-M: -1.0 [5.7] Msed_0927 (hypothetical) -0.3 [0.3] 0.1 [0.1] 0.4 [0.5] none Msed_0928 (hypothetical) 0.2 [0.2] 0.3 [0.4] 0.1 [0.1] YKS-YS: -1.0 [6.5] Msed_0929 (adducin/aldolase) -0.5 [0.6] 0.7 [1.1] 1.2 [2.2] none Msed_0932 (hypothetical) -0.4 [3.1] 0.1 [0.6] 0.5 [4.2] A-H: 3.4 [14.1], A-M: 1.7 [6.6], H-M: -1.7 [6.5] Msed_0934 (hypothetical) 1.0 [4.7] 1.1 [5.0] 0.1 [0.1] none Msed_0935 (aa permease-like) 1.2 [5.2] 1.1 [4.5] -0.1 [0.3] A-H: 3.5 [17.9], A-M: 1.7 [8.3], H-M: -1.8 [8.7] Msed_0936 (hypothetical) 0.1 [0.2] -0.1 [0.1] -0.2 [0.2] none Msed_0937 (hypothetical) -0.6 [0.7] 0.2 [0.2] 0.8 [1.1] none Msed_0938 (hypothetical) -1.2 [2.6] -1.8 [4.6] -0.7 [1.1] H-M: -1.9[ 7.8] Msed_0939 (thioredoxin domain) -0.4 [0.6] 0.8 [1.4] 1.3 [2.6] none Msed_0940 (hypothetical) -0.1 [0.2] 0.9 [6.3] 1.0 [6.8] A-H: 2.1 [13.6], A-M: 1.4 [8.9], Msed_0941 (abortive infection) -0.6 [1.0] -0.6 [1.0] 0.0 [0.0] none Msed_0942 (hypothetical) -0.3 [0.6] -0.3 [0.8] 0.0 [0.1] none Msed_0943 (hypothetical) -1.1 [2.2] -0.6 [1.0] 0.5 [0.7] none a Significance = -log10(p-value). For reference, a significance value of 5.4 correlates to a p-value of 4 x 10-6. b Upregulation reported for H, A, or M conditions represents an average of log2 fold changes and significance values for all 4 combinations of biological repeats (i.e. average of log fold changes for autotrophy vs heterotrophy averages values for individual comparisons A1-H1, A1-H2, A2-H1, and A2-H2)

214